Methods for producing and purifying recombinant alpha-L-iduronidase

ABSTRACT

The present invention provides a recombinant human α-L-iduronidase and biologically active fragments and muteins thereof with a purity greater than 99%. The present invention further provides large-scale methods to produce and purify commercial grade recombinant human α-L-iduronidase enzyme thereof.

[0001] This application claims priority to U.S. application Ser. No.09/711,202, filed Nov. 9, 2000, which is a continuation-in-part of U.S.patent application Ser. No. 09/439,923, filed Nov. 12, 1999.

FIELD OF THE INVENTION

[0002] The present invention is in the field of molecular biology,enzymology, biochemistry and clinical medicine. In particular, thepresent invention provides a human recombinant α-L-iduronidase, methodsof large-scale production and purification of commercial grade humanrecombinant α-L-iduronidase enzyme, and methods to treat certain geneticdisorders including α-L-iduronidase deficiency and mucopolysaccharidosisI (MPS I).

BACKGROUND OF THE INVENTION

[0003] Carbohydrates play a number of important roles in the functioningof living organisms. In addition to their metabolic roles, carbohydratesare structural components of the human body covalently attached tonumerous other entities such as proteins and lipids (calledglycoconjugates). For example, human connective tissues and cellmembranes comprise proteins, carbohydrates and a proteoglycan matrix.The carbohydrate portion of this proteoglycan matrix provides importantproperties to the body's structure.

[0004] A genetic deficiency of the carbohydrate-cleaving, lysosomalenzyme α-L-iduronidase causes a lysosomal storage disorder known asmucopolysaccharidosis I (MPS I) (Neufeld and Muenzer, pp. 1565-1587, inThe Metabolic Basis of Inherited Disease, Eds., C. R. Scriver, A. L.Beaudet, W. S. Sly, and D. Valle, McGraw-Hill, New York (1989)) In asevere form, MPS I is commonly known as Hurler syndrome and isassociated with multiple problems such as mental retardation, cloudingof the cornea, coarsened facial features, cardiac disease, respiratorydisease, liver and spleen enlargement, hernias, and joint stiffness.Patients suffering from Hurler syndrome usually die before age 10. In anintermediate form known as Hurler-Scheie syndrome, mental function isgenerally not severely affected, but physical problems may lead to deathby the teens or twenties. Scheie syndrome is the mildest form of MPS I.It is compatible with a normal life span, but joint stiffness, cornealclouding and heart valve disease cause significant problems.

[0005] The frequency of MPS I is estimated to be 1:100,000 according toa British Columbia survey of all newborns (Lowry, et al., Human Genetics85:389-390 (1990)) and 1:70,000 according to an Irish study (Nelson,Human Genetics 101:355-358 (1990)). There appears to be no ethnicpredilection for this disease. It is likely that worldwide the diseaseis underdiagnosed either because the patient dies of a complicationbefore the diagnosis is made or because the milder forms of the syndromemay be mistaken for arthritis or missed entirely. Effective newbornscreening for MPS I would likely find some previously undetectedpatients.

[0006] Except for a few patients which qualify for bone marrowtransplantation, there are no significant therapies available for allMPS I patients. Hobbs, et al. (Lancet 2: 709-712 (1981)) first reportedthat bone marrow transplantation successfully treated a Hurler patient.Since that time, clinical studies at several transplant centers haveshown improvement in physical disease and slowing or stabilizing ofdevelopmental decline if performed early. (Whitley, et al, Am. J Med.Genet. 46: 209-218 (1993); Vellodi, et al., Arch. Dis. Child. 76: 92-99(1997); Peters, et al., Blood 91: 2601-2608 (1998); Guffon, et al., JPediatrics 133: 119-125 (1998)) However, the significant morbidity andmortality, and the need for matched donor marrow, limits the utility ofbone marrow transplants. An alternative therapy available to allaffected patients, would provide an important breakthrough in treatingand managing this disease.

[0007] Enzyme replacement therapy has been considered a potentialtherapy for MPS I following the discovery that α-L-iduronidase cancorrect the enzymatic defect in Hurler cells in culture, but thedevelopment of human therapy has been technically unfeasible until now.In the corrective process, the enzyme containing a mannose-6-phosphateresidue is taken up into cells through receptor-mediated endocytosis andtransported to the lysosomes where it clears the stored substrates,heparan sulfate and dermatan sulfate. Application of this therapy tohumans has previously not been possible due to inadequate sources ofα-L-iduronidase in tissues.

[0008] For α-L-iduronidase enzyme therapy in MPS I, a recombinant sourceof enzyme has been needed in order to obtain therapeutically sufficientsupplies of the enzyme. The cDNA for the canine enzyme was cloned in1991 (Stoltzfus, et al., J. Biol. Chem. 267:6570-6575 (1992) and for thehuman enzyme in the same year. (Scott, et al., Proc. Natl. Acad. Sci.U.S.A. 88:9695-9699 (1991), Moskowitz, et al., FASEB J 6:A77 (1992)).Following the cloning of cDNA for α-L-iduronidase, the production ofadequate quantities of recombinant α-L-iduronidase allowed the study ofenzyme replacement therapy in canine MPS I. (Kakkis, et al., ProteinExpr. Purif. 5: 225-232 (1994)) Enzyme replacement studies in the canineMPS I model demonstrated that intravenously-administered recombinantα-L-iduronidase distributed widely and reduced lysosomal storage frommany tissues. (Shull, et al., Proc. Natl. Acad. Sci. U.S.A. 91:12937-12941 (1994); Kakkis, et al., Biochem. Mol. Med. 58: 156-167(1996))

BRIEF SUMMARY OF THE INVENTION

[0009] In one aspect, the present invention features a method to massproduce human recombinant α-L-iduronidase in large scale amounts withappropriate purity to enable large scale production for long termpatient use of the enzyme therapy. In a broad embodiment, the methodcomprises the step of transfecting a cDNA encoding for all or part of anα-L-iduronidase into a cell suitable for the expression thereof. In someembodiments, a cDNA encoding for a complete α-L-iduronidase is used,preferably a human α-L-iduronidase. However, in other embodiments, acDNA encoding for a biologically active fragment or mutein thereof maybe used. Specifically, one or more amino acid substitutions may be madewhile preserving or enhancing the biological activity of the enzyme. Inother preferred embodiments, an expression vector is used to transferthe cDNA into a suitable cell or cell line for expression thereof. Inone particularly preferred embodiment, the cDNA is transfected into aChinese hamster ovary cell to create cell line 2.131. In yet otherpreferred embodiments, the production procedure features one or more ofthe following characteristics which have demonstrated particularly highproduction levels: (a) the pH of the cell growth culture may be loweredto about 6.5 to 7.0, preferably to about 6.8-7.0 during the productionprocess, (b) as many as 2 to 3.5 culture volumes of the medium may bechanged during each 24-hour period by continuous perfusion, (c) oxygensaturation may be optimized to about 40% but may be as high as 80%, (d)macroporous cellulose microcarriers with about 5% serum in the mediuminitially, may be used to produce cell mass followed by a rapid washoutshift to protein-free medium for production, (e) a protein-free or lowprotein-medium such as a JRH Biosciences PF-CHO product may be optimizedto include supplemental amounts of one or more ingredients selected fromthe group consisting of: glutamate, aspartate, glycine, ribonucleosides,and deoxyribonucleosides; (f) a stirred tank suspension culture may beperfused in a continuous process to produce iduronidase.

[0010] In a second aspect, the present invention provides a transfectedcell line which features the ability to produce α-L-iduronidase inamounts which enable using the enzyme therapeutically. In preferredembodiments, the present invention features a recombinant Chinesehamster ovary cell line such as the 2.131 cell line that stably andreliably produces amounts of α-L-iduronidase which enable using theenzyme therapeutically. In some preferred embodiments, the cell line maycontain more than 1 copy of an expression construct. In even morepreferred embodiments, the cell line expresses recombinantα-L-iduronidase in amounts of at least 20 micrograms per 10⁷ cells perday.

[0011] In a third aspect, the present invention provides novel vectorssuitable to produce

[0012] α-L-iduronidase in amounts which enable using the enzymetherapeutically. In preferred embodiments, the present inventionfeatures an expression vector comprising a cytomegaloviruspromoter/enhancer element, a 5′ intron consisting of a murine Cα intron,a cDNA encoding all or a fragment or mutein of an α-L-iduronidase, and a3′ bovine growth hormone polyadenylation site. Also, preferably the cDNAencoding all or a fragment or mutein α-L-iduronidase is about 2.2 kb inlength. This expression vector may be transfected at, for example, a 50to 1 ratio with any appropriate common selection vector such as pSV2NEO,to enhance multiple copy insertions. Alternatively, gene amplificationmay be used to induce multiple copy insertions.

[0013] In a fourth aspect, the present invention provides novelα-L-iduronidase produced in accordance with the methods of the presentinvention and thereby present in amounts which enable using the enzymetherapeutically. The specific activity of the α-L-iduronidase accordingto the present invention is in excess of 200,000 units per milligramprotein. Preferably, it is in excess of about 240,000 units permilligram protein. The molecular weight of the α-L-iduronidase of thepresent invention is about 82,000 daltons, about 70,000 daltons beingamino acid, and about 12,000 daltons being carbohydrates.

[0014] In a fifth aspect, the present invention features a novel methodto purify α-L-iduronidase. According to a first embodiment, a cell massmay be grown in about 5% serum-containing medium, followed by a switchto a modified protein-free production medium without any significantadaptation to produce a high specific activity starting material forpurification. In one preferred embodiment, a three step columnchromatography may be used to purify the enzyme. Such a three stepcolumn chromatography may include using a blue sepharose FF, a Cu++chelating sepharose chromatography and a phenyl sepharose HPchromatography. In another preferred embodiment, an acid pH treatmentstep is used to inactivate potential viruses without harming the enzyme.Concanavalin A-Sepharose, Heparin-Sepharose and Sephacryl 200 columnsare removed and Blue-Sepharose and copper chelating columns added toincrease the capacity of the large scale purification process, to reduceundesirable leachables inappropriate for long term patient use, and toimprove the purity of the product.

[0015] In a sixth aspect, the present invention features novel methodsof treating diseases caused all or in part by a deficiency inα-L-iduronidase. In one embodiment, this method features administering arecombinant α-L-iduronidase or a biologically active fragment or muteintherof alone or in combination with a pharmaceutically suitable carrier.In other embodiments, this method features transferring a nucleic acidencoding all or a part of an α-L-iduronidase into one or more host cellsin vivo. Preferred embodiments include optimizing the dosage to theneeds of the organism to be treated, preferably mammals or humans, toeffectively ameliorate the disease symptoms. In preferred embodiments,the disease is Mucopolysaccharidosis I (MPS I), Hurler syndrome,Hurler-Scheie syndrome or Scheie syndrome.

[0016] In a seventh aspect, the present invention features novelpharmaceutical compositions comprising α-L-iduronidase useful fortreating a disease caused all or in part by a deficiency inα-L-iduronidase. Such compositions may be suitable for administration ina number of ways such as parenteral, topical, intranasal, inhalation ororal administration. Within the scope of this aspect are embodimentsfeaturing nucleic acid sequences encoding all or a part of anα-L-iduronidase which may be administered in vivo into cells affectedwith an α-L-iduronidase deficiency.

DESCRIPTION OF THE FIGURES

[0017]FIG. 1 represents the nucleotide and deduced amino acid sequencesof cDNA encoding α-L-iduronidase (SEQ ID NOS:1 and 2). Nucleotides 1through 6200 are provided. Amino acids are provided starting with thefirst methionine in the open reading frame.

[0018]FIG. 2 represents the results from SDS-PAGE runs of eluateobtained according to the procedures as described below. The top panelshows the SDS-PAGE results of purified α-L-iduronidase (3 micrograms)and contaminants from the production/purification scheme disclosed inKakkis, et al., Protein Expr. Purif. 5: 225-232 (1994). In the bottompanel, SDS-PAGE results of purified α-L-iduronidase with contaminantsfrom an unpublished prior production/purification process (U.S. patentapplication Ser. Nos. 09/078,209 and 09/170,977) referred to as theCarson method in Lanes 2 (7.5 microgram α-L-iduronidase) and Lane 3 (5.0microgram α-L-iduronidase) are compared to that of theproduction/purification process of the present invention referred to asthe Galli Process (Lane 4 5 micrograms α-L-iduronidase). Lane 1 containsthe molecular weight marker. FIG. 2 shows that the Galliproduction/purification method of the present invention yields a highlypurified α-L-iduronidase product with fewer contaminants in comparisonwith prior production/purification schemes.

[0019]FIG. 3 demonstrates the α-iduronidase production level over a30-day period, during which time cells are switched at day 5 from aserum—containing medium to a serum-free medium. α-Iduronidase productionwas characterized by: (1) absence of a need for adaptation when cellsare switched from serum-containing to serum-free medium at 100200 (topand bottom panels) with an uninterrupted increase in productivity (toppanel); (2) a high level of production in excess of 4 mg per liter (1000per mL) in a protein-free medium (bottom panel); and (3) a boost inα-iduronidase production with butyrate induction events (bottom panel).

[0020]FIG. 4 demonstrates a decrease in liver volume during enzymetherapy in MPS I patients.

[0021]FIG. 5 demonstrates urinary GAG excretion during enzyme therapy.

[0022]FIG. 6 demonstrates elbow and knee extension in HAC002 duringenzyme therapy.

[0023]FIG. 7 demonstrates shoulder flexion to 104 weeks in four patientswith the most restriction during enzyme therapy.

[0024]FIG. 8 demonstrates improvement in sleep apnea before and aftersix weeks of therapy.

[0025]FIG. 9 demonstrates the improvement in apneas and hypopneas duringsleep with enzyme therapy in each individual patient.

[0026]FIG. 10 demonstrates the improvement in pulmonary function testsbefore and after 12 and 52 weeks of enzyme therapy in one patient.

[0027]FIG. 11 demonstrates increased height growth velocity with enzymetherapy.

[0028]FIG. 12 shows the degree of contamination by Chinese Hamster OvaryProtein (CHOP) and degree of purity of α-L-iduronidase, produced by (1)the Carson method, an unpublished prior production/purification process(U.S. patent application Ser. Nos. 09/078,209 and 09/170,977 and (2) theGalli method, the production/purification process of the presentinvention. Thus, FIG. 12 shows that α-L-iduronidase produced andpurified by the Galli method has a higher degree of purity and lowerdegree of CHOP contamination in comparison to that of the Carson method.

[0029]FIG. 13 shows a comparison of α-L-iduronidase produced by theGalli method versus the Carson method. On the left side of the Figure,results from a Western Blot show that the Galli material (left side,column 2) comprise fewer contaminating protein bands (between 48 kDa and17 kDa) in comparison with the Carson material (left side, column 3). Onthe right side of the Figure, results from an SDS-PAGE silver stain showthe absence of a band at the 62 kDa in the Galli material (column 2) incomparison to the presence of such a band in the Carson material (column3).

DETAILED DESCRIPTION OF THE INVENTION

[0030] In one aspect, the present invention features a method to produceα-L-iduronidase in amounts which enable using the enzymetherapeutically. In general, the method features transforming a suitablecell line with the cDNA encoding for all of α-L-iduronidase or abiologically active fragment or mutein thereof. Those of skill in theart may prepare expression constructs other than those expresslydescribed herein for optimal production of α-L-iduronidase in suitablecell lines transfected therewith. Moreover, skilled artisans may easilydesign fragments of cDNA encoding biologically active fragments andmuteins naturally occurring α-L-iduronidase which possess the same orsimilar biological activity to the naturally occurring full-lengthenzyme.

[0031] To create a recombinant source for α-L-iduronidase, a largeseries of expression vectors may be constructed and tested forexpression of a α-L-iduronidase cDNA. Based on transient transfectionexperiments, as well as stable transfections, an expression constructmay be identified that provides a particularly high level of expression.In one embodiment of the present invention, a Chinese hamster cell line2.131 developed by transfection of the α-L-iduronidase expressionconstruct and selection for a high expression clone providesparticularly high level expression. Such a Chinese hamster cell lineaccording to this embodiment of the present invention may secrete about5,000 to 7,000 fold more α-L-iduronidase than normal. Theα-L-iduronidase produced thereby may be properly processed, taken upinto cells with high affinity and is corrective for α-L-iduronidasedeficient cells, such as those from patients suffering from Hurler'sSyndrome.

[0032] The method for producing α-L-iduronidase in amounts that enableusing the enzyme therapeutically features a production processspecifically designed to mass produce commercial grade enzyme, whereinthe quality of the enzyme has been deemed acceptable for administrationto humans by regulatory authorities of various countries. The largescale production of commercial grade enzyme necessitates modificationsof the cell culture scale, microcarrier systems, and purificationscheme. In preferred embodiments, the cell culture scale is increasedfrom 45 liters to 110 liters or more, with a change to continuousperfusion. The increase in scale is necessary to produce sufficientmaterial for potential large scale production for long term patient use.According to preferred embodiments of such a process, microcarriers areused as a low cost scalable surface on which to grow adherent cells. Inparticularly preferred embodiments, such microcarriers are macroporousand are specifically composed of modified carbohydrates such ascellulose, e.g., Cytopore beads manufactured by Pharmacia. Macroporouscellulose microcarriers allow improved cell attachment and provide alarger surface area for attachment, which is expected to yield anincreased cell density during the culture process. Higher cell densitiesare expected to increase productivity. In preferred embodiments,heparin-Sepharose and Sephacryl 200 columns are replaced withBlue-Sepharose and Copper chelating columns to increase the capacity ofthe large scale purification process and to improve the purity of theproduct. In a particularly preferred embodiment, the copper chelatingcolumn is used to reduce Chinese hamster ovary cell protein contaminantsto very low levels appropriate for large scale distribution. Usingembodiments of the present method featuring modifications and inductiondescribed below, approximately 15 mg per liter of culture per day, ormore at peak culturing density can be produced starting with a 110 literculture system.

[0033] According to other preferred embodiments of the method forproducing α-L-iduronidase according to the present invention, a culturesystem is optimized. In a first embodiment, the culture pH is lowered toabout 6.5 to 7.0, preferably to about 6.7-7.0 during the productionprocess. One advantage of such a pH is to enhance accumulation oflysosomal enzymes that are more stable at acidic pH. In a secondembodiment, as many as 2 to 3.5 culture volumes of the medium may bechanged during each 24-hour period by continuous perfusion. Oneadvantage of this procedure is to enhance the secretion rate ofrecombinant α-L-iduronidase and to capture more active enzyme. In athird embodiment, oxygen saturation is optimized at about 40%. In afourth embodiment, macroporous microcarriers with about 5% seruminitially in the medium, are used to produce a cell mass followed by arapid washout shift to a protein-free medium for production (FIG. 3). Ina fifth embodiment, a protein-free growth medium, such as a JRHIBiosciences PF-CHO product, may be optimized to include supplementalamounts of one or more ingredients selected from the group consistingof: glutamate, aspartate, glycine, ribonucleosides anddeoxyribonucleosides. In a sixth embodiment, as many as 2 to 3.5 culturevolumes of the medium may be changed during each 24-hour period bycontinuous perfusion. Such an induction process may provide about atwo-fold increase in production without significantly alteringpost-translational processing.

[0034] Particularly preferred embodiments of the method for producingα-L-iduronidase according to the present invention feature one, morethan one, or all of the optimizations described herein and may beemployed as described in more detail below. The production method of thepresent invention may, therefore, provide a production culture processhaving the following features:

[0035] 1. A microcarrier based culture using macroporous microcarrierbeads made of modified cellulose or an equivalent thereof is preferablyused in large scale culture flasks with overhead stirring or anequivalent thereof. Attachment of cells to these beads may be achievedby culture in a 5% fetal bovine serum may be added to DME/F12 1:1 or aprotein-free medium modified with ingredients including ribonucleosides,deoxyribonucleosides, pyruvate, non-essential amino acids, and HEPES.After about 3-6 days in this medium, a washout procedure is begun inwhich protein-free medium replaces the serum-containing medium at anincreasing perfusion rate dependent on the glucose content and culturecondition. Subsequently, and throughout the entire remaining cultureperiod, the cells are cultivated in a protein-free medium. The use of aprotein-free medium in enzyme production is beneficial in reducing theexposure risk of bovine spongiform encephalopathy (BSE) and otherinfectious biologic agents such as viruses to patients being treatedwith the enzyme, wherein the risk of BSE or other harmful agents isdependent on the amount of potential serum exposure. In prior publishedstudies, the carriers used to grow the cells were bovine gelatinmicrocarriers, used at 1 gram per liter or 100 times the productconcentration. Leaching of 1% of the gelatin protein from themicrocarriers would represent a relative 100% contamination and therebycontribute to the risk of BSE. Thus, new carriers are either dextran orcellulose-based and consist of carbohydrates, and not animal-derivedmaterials.

[0036]FIG. 3 shows that the cells are grown to a density in 5% serumcontaining medium and then switched without any adaptation to aprotein-free medium. FIG. 3 specifically shows that: 1) Cells surviveand continue to produce iduronidase when shifted without adaptation. Incontrast, other studies would suggest that adaptation to a protein-freemedium is necessary. In the method of the present invention, enzymeproduction continues at levels comparable to serum containing medium. 2)α-L-Iduronidase produced in a protein-free medium retains a level ofproduction in excess of 4 mg per liter or 1,000 units per ml. 3)α-L-Iduronidase produced in a protein-free medium has high uptakeindicating that the shift in medium and, hence, a shift in carbohydratesbeing fed to cells, does not adversely affect the high uptake characterof the enzyme. Eight lots of α-L-iduronidase have been produced andreleased in this manner with an uptake half maximal value of less than 2nM in all lots.

[0037] 2. The culture conditions are preferably maintained at adissolved oxygen of 40% of air saturation at a pH of about 6.8-7.0 andat a temperature of about 35-37° C. This may be achieved using a controlunit, monitoring unit and appropriate probes such as those produced byApplikon® or Mettler®. However, skilled artisans will readily appreciatethat this can easily be achieved by equivalent control systems producedby other manufacturers. An air saturation of about 40% results inimproved α-L-iduronidase secretion though up to 80%% air saturation maybe used. However, further increases in oxygen to, for example, 90% airsaturation, do not provide significantly enhanced secretion over 80% airsaturation. The dissolved oxygen may be supplied by intermittent orcontinuous oxygen sparging using a 5 micron stainless steel or largeropening sparger, or equivalent thereof. A pH of about 6.8-7.0 is optimalfor the accumulation of the α-L-iduronidase enzyme. The enzyme isparticularly unstable at pHs above about 7.0. Below a pH of about 6.7,the secretion rate may decrease, particularly below a pH of about 6.5.The culture is therefore maintained optimally between a pH of about6.8-7.0.

[0038] 3. The production culture medium may be a modified form of thecommercially available proprietary medium from JRH Biosciences calledExcell PF CHO. This medium supports levels of secretion equivalent tothat of serum using a cell line such as the 2.131 cell line. It may bepreferably modified to include an acidic pH of about 6.8-7.0 (±0.1), andbuffered with HEPES at 7.5 mM or 15 mM. The medium may contain 0.05 to0.1% of Pluronics F-68 (BASF), a non-ionic surfactant or an equivalentthereof which features the advantage of protecting cells from shearforces associated with sparging. The medium may further contain aproprietary supplement that is important in increasing the productivityof the medium over other protein-free media that are presentlyavailable. Those skilled in the art will readily understand that thechoice of culture medium may be optimized continually according toparticular commercial embodiments available at particular points intime. Such changes encompass no more than routine experimentation andare intended to be within the scope of the present invention.

[0039] 4. The production medium may be analyzed using an amino acidanalyzer comparing spent medium with starting medium. Such analyses havedemonstrated that the 2.131 cell line depletes a standard PF CHO mediumof glycine, glutamate and aspartate to a level of around 10% of thestarting concentration. Supplementation of these amino acids to higherlevels may result in enhanced culture density and productivity that maylead to a 2-3 fold higher production than at baseline. Skilled artisanswill appreciate that other cell lines within the scope of the presentinvention may be equally useful for producing α-L-iduronidase accordingto the present method. Hence, more or less supplemental nutrients may berequired to optimize the medium. Such optimizations are intended to bewithin the scope of the present invention and may be practiced withoutundue experimentation.

[0040] 5. The medium may be supplemented with the four ribonucleosidesand four deoxyribonucleosides each at about 10 mg/liter to support thedihydrofolate reductase deficient cell line 2.131. Skilled artisans willappreciate that other cell lines within the scope of the presentinvention may be equally useful for producing α-L-iduronidase accordingto the present method. Hence, more or less ribonucleosides anddeoxyribonucleosides may be required to optimize the medium, andalternative sources of purines and pyrmidines for nucleic acid synthesismay be used such as hypoxanthine and thymidine. Such optimizations areintended within the scope of the present invention and may be practicedwithout undue experimentation.

[0041] 6. After reaching confluence at about 3-6 days of culture, anincreasing rate of continuous perfusion is initiated. A change of mediummay be accomplished, for example, using a slant feed tube constructedand positioned to allow the uptake of medium without removal of themicrocarriers even while the culture is stirred. By pumping out mediumthrough the slant feed tube, microcarriers settle within the body of thetube inside the culture and are not removed from the culture during thechange on medium. In this manner, the microcarriers with the cell massare separated from supernatant containing the enzyme.

[0042] 7. The rapid and frequent turnover of the medium has been shownby productivity studies to result in improved overall collection ofenzyme from the cell culture. Less turnover of medium results in lesstotal production of enzyme on a daily basis. Using the perfusion of2-3.5 culture volumes per day, the cells may be maintained in excellentcondition with high degrees of viability and a high level ofproductivity.

[0043] 8. Production of α-L-iduronidase may be enhanced by the use ofsodium butyrate induction of gene expression (FIG. 3). Twenty lots ofα-L-iduronidase were produced using butyrate induction at 2 nMconcentration with ⅔ washout every 12 hours after induction andreinduction every 48 hours for a 21-day production period. In FIG. 3,the vertical arrows at the bottom indicate butyrate induction events.Each induction triggered a boost in α-L-iduronidase concentration in themedium.

[0044] Systematic studies of a 2.131 cell line demonstrated that about 2mM butyrate can be applied and result in about a two-fold or greaterinduction of enzyme production with minimal effects on carbohydrateprocessing. Lower levels of butyrate have not been shown to induce aswell, and substantially higher levels may result in higher induction,but declining affinity of the produced enzyme for cells from patientssuffering from α-L-iduronidase deficiency. Butyrate induction performedin vitro at 2 mM for 24 hours or 5 mM, a more commonly usedconcentration resulted in uptakes in excess of 3 nM or 40 U/ml, or anaverage of three times the value observed in production lots. Inaddition, commonly used times of 24 hours or more and concentration of 5mM were toxic to α-L-iduronidase producing cells and resulted indetachment and loss of cell mass.

[0045] Results suggest that two-fold or greater induction results inless processing of the carbohydrates and less phosphate addition to theenzyme, as well as increasing toxicity. With respect to carbohydrateprocessing and the addition of phosphate groups, the importance ofmannose-6-phosphate in enzyme replacement therapy is demonstrated by theobservations that removal of the phosphate of two lysosomal enzymes,glucosidase and galactosamine 4-sulfatase leads to decreased uptake (Vander Ploeg, et al., J. Clin. Invest. 87: 513-518 (1991); Crawley, et al.,J. Clin. Invest. 97: 1864-1873 (1996)). In addition, enzyme with lowphosphate (Van Hove, et al., Proc. Natl. Acad. Sci. USA 93: 65-70 (1996)requires 1,000 units per ml for uptake experiments (nearly 100 timesused for iduronidase) and effective doses in animal models require 14mg/kg, or 28 times the dose used with high phosphate containingiduronidase (Kikuchi, et al., J. Clin. Invest. 101: 827-833 (1998)).

[0046] One particularly preferred aspect of the invention method uses 2mM butyrate addition every 48 hours to the culture system. Thisembodiment results in about a two-fold induction of enzyme productionusing this method without significant effect on the uptake affinity ofthe enzyme (K-uptake of less than 30 U/ml or 2.0 mM).

[0047] In a second aspect, the present invention provides a transfectedcell line, which possesses the unique ability to produce α-L-iduronidasein amounts, which enable using the enzyme therapeutically. In preferredembodiments, the present invention features a recombinant Chinesehamster ovary cell line such as the 2.131 cell line that stably andreliably produces amounts of α-L-iduronidase. In preferred embodiments,the cell line may contain more than 1 copy of an expression constructcomprising a CMV promoter, a Cα intron, a human α-L-iduronidase cDNA,and a bovine growth hormone polyadenylation sequence. In even morepreferred embodiments, the cell line expresses α-L-iduronidase atamounts of at least about 20-40 micrograms per 10⁷ cells per day in aproperly processed, high uptake form appropriate for enzyme replacementtherapy. According to preferred embodiments of this aspect of theinvention, the transfected cell line adapted to produce α-L-iduronidasein amounts which enable using the enzyme therapeutically, possesses oneor more of the following features:

[0048] 1. The cell line of preferred embodiments is derived from aparent cell line wherein the cells are passaged in culture until theyhave acquired a smaller size and more rapid growth rate and until theyreadily attach to substrates.

[0049] 2. The cell line of preferred embodiments is transfected with anexpression vector containing the cytomegalovirus promoter/enhancerelement, a 5′ intron consisting of the murine Cα intron between exons 2and 3, a human cDNA of about 2.2 kb in length, and a 3′ bovine growthhormone polyadenylation site. This expression vector may be transfectedat, for example, a 50 to 1 ratio with any appropriate common selectionvector such as pSV2NEO. The selection vector pSV2NEO in turn confersG418 resistance on successfully transfected cells. In particularlypreferred embodiments, a ratio of about 50 to 1 is used since this ratioenhances the acquisition of multiple copy number inserts. According toone embodiment wherein the Chinese hamster ovary cell line 2.131 isprovided, there is at least 1 copy of the expression vector forα-L-iduronidase. Such a cell line has demonstrated the ability toproduce large quantities of human α-L-iduronidase (minimum 20 microgramsper 10 million cells per day). Particularly preferred embodiments suchas the 2.131 cell line possess the ability to produce properly processedenzyme that contains N-linked oligosaccharides containing high mannosechains modified with phosphate at the 6 position in sufficient quantityto produce an enzyme with high affinity (K-uptake of less than 3 nM).

[0050] 3. The enzyme produced from the cell lines of the presentinvention such as a Chinese hamster ovary cell line 2.131 is rapidlyassimilated into cells, eliminates glycosaminoglycan storage and has ahalf-life of about 5 days in cells from patients suffering fromα-L-iduronidase deficiency.

[0051] 4. The cell line of preferred embodiments such as a 2.131 cellline adapts to large scale culture and stably produces humanα-L-iduronidase under these conditions. The cells of preferredembodiments are able to grow and secrete α-L-iduronidase at the acid pHof about 6.6 to 7.0 at which enhanced accumulation of α-L-iduronidasecan occur.

[0052] 5. Particularly preferred embodiments of the cell line accordingto the invention, such as a 2.131 cell line are able to secrete humanα-L-iduronidase at levels exceeding 2,000 units per ml (8 micrograms perml) harvested twice per day or exceeding 15 mg per liter of culture perday using a specially formulated protein-free medium.

[0053] In a third aspect, the present invention provides novel vectorssuitable to produce α-L-iduronidase in amounts which enable using theenzyme therapeutically. The production of adequate quantities ofrecombinant α-L-iduronidase is a critical prerequisite for studies onthe structure of the enzyme as well as for enzyme replacement therapy.The cell lines according to the present invention permit the productionof significant quantities of recombinant α-L-iduronidase that isappropriately processed for uptake. Overexpression in Chinese hamsterovary (CHO) cells has been described for three other lysosomal enzymes,α-galactosidase (Ioannou, et al., J Cell. Biol. 119:1137-1150 (1992)),iduronate 2-sulfatase (Bielicki, et al., Biochem. J. 289: 241-246(1993)), and N-acetylgalactosamine 4-sulfatase (Amson, et al., Biochem.J. 284:789-794 (1992)), using a variety of promoters and, in one case,amplification. The present invention features a dihydrofolatereductase-deficient CHO cell line, but according to preferredembodiments of the invention amplification is unnecessary. Additionally,the present invention provides a high level of expression of the humanα-L-iduronidase using the CMV immediate early gene promoter/enhancer.

[0054] The present invention features in preferred embodiments, anexpression vector comprising a cytomegalovirus promoter/enhancerelement, a 5′ intron consisting of the murine Cα intron derived from themurine long chain immunoglobulin Cα gene between exons 2 and 3, a humancDNA of about 2.2 kb in length, and a 3′ bovine growth hormonepolyadenylation site. This expression vector may be transfected at, forexample, a 50 to 1 ratio with any appropriate common selection vectorsuch as pSV2NEO. The selection vector such as pSV2NEO in turn confersG418 resistance on successfully transfected cells. In particularlypreferred embodiments, a ratio of about 50 to 1 expression vector toselection vector is used since this ratio enhances the acquisition ofmultiple copy number inserts. According to one embodiment wherein theChinese hamster ovary cell line 2.131 is provided, there areapproximately 10 copies of the expression vector for α-L-iduronidase.Such an expression construct has demonstrated the ability to producelarge quantities of human α-L-iduronidase (minimum 20 micrograms per 10million cells per day) in a suitable cell line such as a Chinese hamsterovary cell line 2.131.

[0055] In a fourth aspect, the present invention provides novelα-L-iduronidase produced in accordance with the methods of the presentinvention and thereby present in amounts that enable using the enzymetherapeutically. The methods of the present invention produce asubstantially pure α-L-iduronidase that is properly processed and inhigh uptake form, appropriate for enzyme replacement therapy andeffective in therapy in vivo.

[0056] The specific activity of the ac-L-iduronidase according to thepresent invention is in excess of about 200,000 units per milligramprotein. Preferably, it is in excess of about 240,000 units permilligram protein using the original assay methods for activity andprotein concentration. A novel validated assay for the same enzyme withunits expressed as micromoles per min demonstrates an activity of 100units/ml (range of 70-130) and a protein concentration by absorbance at280 nM of 0.7 mg/ml (0.6-0.8) with an average specific activity of 143units per mg. The molecular weight of the full length α-L-iduronidase ofthe present invention is about 82,000 daltons comprising about 70,000daltons of amino acids and 12,000 daltons of carbohydrates. Therecombinant enzyme of the present invention is endocytosed even moreefficiently than has been previously reported for a partially purifiedpreparation of urinary enzyme. The recombinant enzyme according to thepresent invention is effective in reducing the accumulation ofradioactive S-labeled GAG in α-L-iduronidase-deficient fibroblasts,indicating that it is transported to lysosomes, the site of GAG storage.The remarkably low concentration of α-L-iduronidase needed for suchcorrection (half-maximal correction at 0.7 pM) may be very important forthe success of enzyme replacement therapy.

[0057] The human cDNA of α-L-iduronidase predicts a protein of 653 aminoacids and an expected molecular weight of 70,000 daltons after signalpeptide cleavage. Amino acid sequencing reveals alanine 26 at theN-terminus giving an expected protein of 629 amino acids. Humanrecombinant α-L-iduronidase has a Histidine at position 8 of the matureprotein. The predicted protein sequence comprises six potential N-linkedoligosaccharide modification sites. All of these may be modified in therecombinant protein. The third and sixth sites have been demonstrated tocontain one or more mannose 6-phosphate residues responsible for highaffinity uptake into cells. The following peptide corresponds to AminoAcids 26-45 of Human Recombinant α-L-iduronidase with an N-terminusalanine and the following sequence:

[0058]ala-glu-ala-pro-his-leu-val-his-val-asp-ala-ala-arg-ala-leu-trp-pro-leu-arg-arg(art of SEQ ID NO:2)

[0059] The overexpression of the α-L-iduronidase of the presentinvention does not result in generalized secretion of other lysosomalenzymes that are dependent on mannose-6-P targeting. The secretedrecombinant α-L-iduronidase is similar to normal secreted enzyme in manyrespects. Its molecular size, found in various determinations to be 77,82, 84, and 89 kDa, is comparable to 87 kDa, found for urinarycorrective factor (Barton et al., J. Biol. Chem. 246: 7773-7779 (1971)),and to 76 kDa and 82 kDa, found for enzyme secreted by cultured humanfibroblasts (Myerowitz, et al, J. Biol. Chem. 256: 3044-3048 (1991);Taylor, et al., Biochem. J 274:263-268 (1991)). The differences withinand between the studies are attributed to imprecision of themeasurements. The pattern of intracellular processing of the recombinantenzyme, a slow decrease in molecular size and the eventual appearance ofan additional band smaller by 9 kDa is the same as for the humanfibroblast enzyme. This faster band arises by proteolytic cleavage of 80N-terminal amino acids.

[0060] In a fifth aspect, the present invention features a novel methodto purify α-L-iduronidase. The U.S. Food and Drug Administration hasissued recommendations for assembling chemical and technological datacurrently considered appropriate for an enzyme preparation, includingguidelines regarding the purity of enzyme preparations (EnzymePreparations: Chemistry Recommendations for Food Additive and GRAS[Generally Recommended As Safe] Affirmation Petitions, Version 1.1, Jan.23, 1993; U.S. Food and Drug Administration, Center For Food Safety andApplied Nutrition, Office of Premarket Approval, Chemistry ReviewBranch). Various studies have shown that impurities, such asanticomplement activity, in protein preparations, includingimmunoglobulin preparations, may be associated with the development ofallergic and anaphylactic reactions (Lundblad, et al., Rev. Infect. Dis.8 (Suppl. 4):S382-90 (1986); Scheiermann and Kuwert, Dev. Biol. Stand.44:165-171(1979)). Furthermore, impurities may be associated withunwanted biological activities and interference with desired therapeuticeffects. Thus, enhanced purity of protein preparations would contributeto greater efficacy of the therapeutic protein (Ueshima, et al., J.Clin. Hosp. Pharm. 10(2): 193-202 (1985); Ehrlich, et al., Clin. Chem.34(9): 1681-8 (1988)).

[0061] The relationship between enzyme purity and immunogenicity isdemonstrated in Studies 1 (Example 5) and 2 (Example 6). Two types ofimmune reactions, urticaria and complement activation (indicated bylaboratory analysis), were documented during enzyme infusion and may beassociated with enzyme therapy. In the Phase I study (Example 5), thepurity of recombinant human α-L-iduronidase was between 96% to 98%. Inthe Phase III study (Example 6), recombinant human α-L-iduronidase waspurified to greater than 99%. FIGS. 12 and 13 compare the degree ofcontamination by the other proteins, such as Chinese Hamster OvaryProtein, and the purity of the recombinant human α-L-iduronidaseproduced by the previous Carson and current Galli methods. The resultsshow that the recombinant human α-L-iduronidase purified according tothe Galli method has fewer protein contaminants than enzyme produced bythe Carson method. In the Phase I study using enyme purified to 96-98%,five patients developed urticaria and evidence of complement activationwas observed in four patients. In the Phase III study using enzymepurified to greater than 99%, none of the enzyme-treated patientsdeveloped urticaria. Although all enzyme—treated patients seroconvertedin immunogenicity testing for IgG, seroconversion did not result inincreased infusion-associated reactions or other adverse events. Inpatients tested for IgE, results were negative. The relationship betweenpurity and immunogenicity is even more evident in the animal studiesdescribed in Example 3, wherein the purity of the recombinant humanα-L-iduronidase was equal or less than or about 95%. In the animalstudies, all MPS I dogs and most MPS I cats receiving enzyme treatmentdeveloped antibodies, including IgG antibodies of thecomplement-acrivating type, a phenomenon observed in 13% ofalglucerase-treated Gaucher patients. One MPS I dog also developedproteinuria thought to be related to immune complex disease. Thesestudies suggest that an increased level of enzyme purity is associatedwith a lower frequency of immune-related adverse side effects and hencewith greater safety and efficacy of enzyme therapy.

[0062] In preferred embodiments, the present invention features a methodto purify recombinant α-L-iduronidase that has been optimized to producea rapid and efficient purification with validatible chromatographyresins and easy load, wash and elute operation. The method of purifyingα-L-iduronidase of the present invention involves a series of columnchromatography steps, which allow the high yield purification of enzymefrom protein-free production medium. Specifically, ConcanavalinA-Sepharose, Heparin-Sepharose and Sephacryl 200 columns were replacedwith Blue-Sepharose and Copper chelating columns to increase thecapacity of a large-scale purification process, to reduce leachables andto improve the purity of the product. Concanavalin A lectin is oftenused to bind enzyme in an initial purification step in the priorpublished study, and is a protein lectin derived from plants.Concanavalin A is known to leach from columns and contaminate lysosomalenzyme preparations. Such leaching could cause activation of T cells intreated patients and hence is deemed inappropriate for humanadministration (Furbish, et al., Proc. Natl. Acad. Sci. USA 74:3560-3563 (1977)). Thus, the use of Concanavalin A is avoided in thepresent purification scheme. In a prior study, the human liverα-L-iduronidase could not be recovered from phenyl columns without highconcentrations of detergent (1% Triton X100) denaturation. Hence, aphenyl column was not used in a published purification scheme of thisenzyme (Clements, et al., Eur. J. Biochem. 152: 21-28 (1985). Theendogenous human liver enzyme is highly modified within the lysosomes byhydrolases which remove sialic acid and phosphate residues and proteaseswhich nick the enzyme. In contrast, the overexpression of recombinantα-L-iduronidase causes 50% of the enzyme to be secreted rather thantransported to the lysosome (Zhao, et al., J. Biol Chem. 272:22758-22765 (1997). Hence, recombinant iduronidase will have a fullarray of sialic acid and phosphate residues, which lead to a higherdegree of water solubility and lower affinity to the phenyl column. Theincreased hydrophilicity allows the enzyme to be eluted undernon-denaturing conditions using the low salt solutions of around 150-700mM NaCl. This feature of the recombinant enzyme allows it to be purifiedin large scale without the use of detergents.

[0063] Recombinant α-L-iduronidase over-expressed in a Chinese HamsterOvary (CHO) cell line, has been purified to near homogeneity following a3-step column chromatography process. The first column involves anaffinity chromatography step using Blue Sepharose 6 FF. The BlueSepharose 6 FF eluate is then further purified by another affinitychromatography step using Cu⁺⁺ Chelating Sepharose FF. The final polishof the highly purified enzyme is achieved by hydrophobic interactionchromatography using Phenyl Sepharose High Performance (HP). Theover-all yield ranges from 45 to 55 percent and the purity of the finalproduct is >99%. The process is robust, reproducible, and scalable forlarge-scale manufacturing. The purified enzyme has been characterizedwith respect to its enzymatic activity using a fluorescence-basedsubstrate, and its functional uptake by fibroblast cells. The enzyme hasalso been characterized for substrate specificity, carbohydrateprofiles, and isoelectric focusing (IEF) profiles.

[0064] Particularly preferred embodiments of the method for purifyingα-L-iduronidase according to the present invention feature more than oneor all of the optimizations according to the following particularembodiments. The purification method of the present invention maytherefore provide a purified α-L-iduronidase having the characteristicsdescribed herein. Outline of the α-L-Iduronidase Purification Process

↓

↓

↓

↓

↓

[0065] 1. pH Adjustment/Filtration: The pH of filtered harvest fluid(HF) is adjusted to 5.3 with 1 M H₃PO₄ and then filtered through a 0.45μfilter (e.g. Sartoclean, Sartorius).

[0066] 2. Blue Sepharose FF chromatography: This affinity chromatographystep serves to capture iduronidase to reduce the volume and to purifyiduronidase by approximately seven to ten fold. Loading capacity: 4mg/ml (total protein per ml of resin) Equilibration buffer: 10 mM NaPO₄,pH 5.3 Wash buffer: 400 mM NaCl, 10 mM NaPO₄, pH 5.3 Elution buffer: 0.8M NaCl, 10 mM NaPO₄, pH 5.3 Regeneration buffer: 2 M NaCl, 10 mM NaPO₄,pH 5.3 Fold of purification: 7-10 Yield: 70-85%

[0067] 3. Cu⁺⁺ Chelating Sepharose FF chromatography: The Cu⁺⁺ Chelatingaffinity chromatography step is very effective for removing somecontaminating CHO proteins. The inclusion of 10% glycerol in all thebuffers seems to be crucial for the quantitative recovery ofiduronidase. Loading capacity: 2 mg/ml Equilibration buffer: 1 M NaCl,25 mM NaAc, pH 6.0, 10% Glycerol Wash buffer: 1 M NaCl, 25 mM NaAc, pH4.0, 10% Glycerol Elution buffer: 1 M NaCl, 25 mM NaAc, pH 3.7, 10%Glycerol Regeneration buffer: 1 M NaCl, 50 mM EDTA, pH 8.0 Fold ofpurification: 2-5 Yield: 80%

[0068] 4. Phenyl Sephrose HP chromatography: Phenyl Sephrose is used asthe last step to further purify the product as well as to reduceresidual leached Cibacron blue dye and Cu⁺⁺ ion carried over fromprevious columns. Loading capacity: 1 mg/ml Equilibration buffer: 2 MNaCl, 10 mM NaPO₄, pH 5.7 Wash buffer: 1.5 M NaCl, 10 mM NaPO₄, pH 5.7Elution buffer: 0.7 M NaCl, 10 mM NaPO₄, pH 5.7 Regeneration buffer: 0 MNaCl, 10 mM NaPO₄, pH 5.7 Fold of purification: 1.5 Yield: 90%

[0069] 5. Ultrafiltration (UF)/Diafiltration (DF)/Final formulation: Thepurified iduronidase is concentrated and diafiltered to a finalconcentration of 1 mg/ml in formulation buffer (150 mM NaCl, 100 mMNaPO₄, pH 5.8) using a tangential flow filtration (TFF) system (e.g.Sartocon Slice from Sartorius). The enzyme is then sterilized byfiltering through a 0.2-micron filter (e.g., cellulose acetate orpolysulfone) and filled into sterile vials.

[0070] 6. Characterization of Purified Iduronidase: Analysis of enzymepurity using SDS-PAGE stained with Coomassie Blue or Silver and Westernblot analysis. Analysis of enzymatic activity using 4MU-sulfate assubstrate. Analysis of functional uptake using fibroblast cell assay.Analysis of carbohydrates by FACE. Analysis of IEF profiles.

[0071] Enzyme purified in this manner has been shown to containmannose-6-phosphate residues of sufficient quantity at positions 3 and 6of the N-linked sugars to give the enzyme uptake affinity of less than30 units per ml (less than 2 nM) enzyme. The enzyme is substantiallycorrective for glycosaminoglycan storage disorders caused by iduronidasedeficiency and has a half-life inside cells of approximately 5 days.

[0072] Prior α-L-iduronidase purification schemes (Kakkis, et al,Protein Expr. Purif 5: 225-232 (1994); Kakkis, et al., Biochem. Mol.Med. 58: 156-167 (1996); U.S. patent application Ser. Nos. 09/078,209and 09/170,977) produced degrees of purity between 90% and less than99%, which is not optimal for long-term human administration (See FIG.12). (These and all other U.S. patents herein are specificallyincorporated herein by reference in their entirety.) Treatment withhuman recombinant α-L-iduronidase with a minimum purity of 97% wasassociated with some clinical reactions, specifically hives in 5patients, and complement activation in 4 patients. All patientsdemonstrated a reaction to a protein that is a trace contaminant to theα-L-iduronidase. (FIG. 2) Because this protein exists in both the finalproduct and in the serum-free blank CHO cell line supernatant, theextraneous protein most likely originates from the CHO cell. The commonproteins that appear to be activating the clinical allergic response areapproximately 60 kDaltons and 50 kDaltons respectively, which are toosmall to be recombinant human iduronidase. Four patients developed animmune reaction to α-L-iduronidase at least transiently as well as tothe Chinese hamster ovary cell host proteins. It is clear that eventhough the enzyme used to treat patients is highly purified, the degreeof purification is important in reducing the immune response tocontaminants. FIG. 2 (SDS-PAGE), FIG. 12 (CHOP assay), and FIG. 13(Western Blot, Silver Stain) demonstrate that α-L-iduronidase producedand purified by the production/purification scheme of the presentinvention has a higher degree of purity and lower degree of CHOPcontamination in comparison to that of prior methods ofproduction/purification. Thus, a greater than 97% purity is adequate forpatient use, higher levels of purity are desirable and preferable. Asshown in FIG. 12, the optimized purification scheme described aboveachieves a degree of purity that is greater than 99% and importantlyreduces Chinese hamster ovary cell host proteins to less than 1 percent,as determined by the Chinese Hamster Ovary Protein (CHOP) assay.

[0073] In a sixth aspect, the present invention features novel methodsof treating diseases caused all or in part by a deficiency inα-L-iduronidase. Recombinant α-L-iduronidase provides enzyme replacementtherapy in a canine model of MPS 1. This canine model is deficient inα-L-iduronidase due to a genetic mutation and is similar to human MPS 1.Purified, properly processed α-L-iduronidase was administeredintravenously to 11 dogs. In those dogs treated with weekly doses of25,000 to 125,000 units per kg for 0.5, 3, 6 or 13 months, the enzymewas taken up in a variety of tissues and decreased the lysosomal storagein many tissues. The long term treatment of the disease was associatedwith clinical improvement in demeanor, joint stiffness, coat and growth.Higher doses of therapy (125,000 units per kg per week) result in betterefficacy, including normalization of urinary GAG excretion in additionto more rapid clinical improvement in demeanor, joint stiffness andcoat.

[0074] Enzyme therapy at even small doses of 25,000 units (0.1 mg/kg/wk)resulted in significant enzyme distribution to some tissues anddecreases in GAG storage. If continued for over 1 year, some clinicaleffects were evident in terms of increased activity, size and overallappearance of health. The therapy at this dose did not improve othertissues that are important sites for disease in this entity such ascartilage and brain. Higher doses of 125,000 units (0.5 mg/kg) given 5times over two weeks demonstrate that improved tissue penetration can beachieved, and a therapeutic effect at the tissue level was accomplishedin as little as 2 weeks. Studies at this increased dose have beencompleted in two dogs for 15 months. These MPS I dogs are showingsignificant clinical improvement and substantial decreases in urinaryGAG excretion into the near normal range. Other than an immune reactioncontrolled by altered administration techniques, the enzyme therapy hasnot shown significant clinical or biochemical toxicity. Enzyme therapyat this higher weekly dose is effective at improving some clinicalfeatures of MPS I and decreasing storage without significant toxicity.

[0075] In a seventh aspect, the present invention features novelpharmaceutical compositions comprising human α-L-iduronidase useful fortreating a deficiency in α-L-iduronidase. The recombinant enzyme may beadministered in a number of ways such as parenteral, topical,intranasal, inhalation or oral administration. Another aspect of theinvention is to provide for the administration of the enzyme byformulating it with a pharmaceutically acceptable carrier, which may besolid, semi-solid, liquid, or an ingestable capsule. Examples ofpharmaceutical compositions include tablets, drops such as nasal drops,compositions for topical application such as ointments, jellies, creamsand suspensions, aerosols for inhalation, nasal spray, and liposomes.Usually the recombinant enzyme comprises between 0.01 and 99% or between0.01 and 99% by weight of the composition, for example, between 0.01 and20% for compositions intended for injection and between 0.1 and 50% forcompositions intended for oral administration.

[0076] To produce pharmaceutical compositions in this form of dosageunits for oral application containing a therapeutic enzyme, the enzymemay be mixed with a solid, pulverulent carrier, for example lactose,saccharose, sorbitol, mannitol, a starch such as potato starch, cornstarch, amylopectin, laminaria powder or citrus pulp powder, a cellulosederivative or gelatin and also may include lubricants such as magnesiumor calcium stearate or a Carbowax® or other polyethylene glycol waxesand compressed to form tablets or cores for dragees. If dragees arerequired, the cores may be coated for example with concentrated sugarsolutions which may contain gum arabic, talc and/or titanium dioxide, oralternatively with a film forming agent dissolved in easily volatileorganic solvents or mixtures of organic solvents. Dyestuffs can be addedto these coatings, for example, to distinguish between differentcontents of active substance. For the composition of soft gelatincapsules consisting of gelatin and, for example, glycerol as aplasticizer, or similar closed capsules, the active substance may beadmixed with a Carbowax® or a suitable oil, e.g., sesame oil, olive oil,or arachis oil. Hard gelatin capsules may contain granulates of theactive substance with solid, pulverulent carriers such as lactose,saccharose, sorbitol, mannitol, starches such as potato starch, cornstarch or amylopectin, cellulose derivatives or gelatin, and may alsoinclude magnesium stearate or stearic acid as lubricants.

[0077] Therapeutic enzymes of the subject invention may also beadministered parenterally such as by subcutaneous, intramuscular orintravenous injection or by sustained release subcutaneous implant. Insubcutaneous, intramuscular and intravenous injection, the therapeuticenzyme (the active ingredient) may be dissolved or dispersed in a liquidcarrier vehicle. For parenteral administration, the active material maybe suitably admixed with an acceptable vehicle, preferably of thevegetable oil variety such as peanut oil, cottonseed oil and the like.Other parenteral vehicles such as organic compositions using solketal,glycerol, formal, and aqueous parenteral formulations may also be used.

[0078] For parenteral application by injection, compositions maycomprise an aqueous solution of a water soluble pharmaceuticallyacceptable salt of the active acids according to the invention,desirably in a concentration of 0.01-10%, and optionally also astabilizing agent and/or buffer substances in aqueous solution. Dosageunits of the solution may advantageously be enclosed in ampules.

[0079] When therapeutic enzymes are administered in the form of asubcutaneous implant, the compound is suspended or dissolved in a slowlydispersed material known to those skilled in the art, or administered ina device which slowly releases the active material through the use of aconstant driving force such as an osmotic pump. In such cases,administration over an extended period of time is possible.

[0080] For topical application, the pharmaceutical compositions aresuitably in the form of an ointment, gel, suspension, cream or the like.The amount of active substance may vary, for example, between 0.05-20%by weight of the active substance. Such pharmaceutical compositions fortopical application may be prepared in known manner by mixing the activesubstance with known carrier materials such as isopropanol, glycerol,paraffin, stearyl alcohol, polyethylene glycol, etc. Thepharmaceutically acceptable carrier may also include a known chemicalabsorption promoter. Examples of absorption promoters are, e.g.,dimethylacetamide (U.S. Pat. No. 3,472,931), trichloro ethanol ortrifluoroethanol (U.S. Pat. No. 3,891,757), certain alcohols andmixtures thereof (British Patent No. 1,001,949). A carrier material fortopical application to unbroken skin is also described in the Britishpatent specification No. 1,464,975, which discloses a carrier materialconsisting of a solvent comprising 40-70% (v/v) isopropanol and 0-60%(v/v) glycerol, the balance, if any, being an inert constituent of adiluent not exceeding 40% of the total volume of solvent.

[0081] The dosage at which the therapeutic enzyme containingpharmaceutical compositions are administered may vary within a widerange and will depend on various factors such as the severity of thedisease, the age of the patient, etc., and may have to be individuallyadjusted. A possible range for the amount of therapeutic enzyme whichmay be administered per day is about 0.1 mg to about 2000 mg or about 1mg to about 2000 mg.

[0082] The pharmaceutical compositions containing the therapeutic enzymemay suitably be formulated so that they provide doses within theseranges, either as single dosage units or as multiple dosage units. Inaddition to containing a therapeutic enzyme (or therapeutic enzymes),the subject formulations may contain one or more substrates or cofactorsfor the reaction catalyzed by the therapeutic enzyme in thecompositions. Therapeutic enzymes containing compositions may alsocontain more than one therapeutic enzyme.

[0083] The recombinant enzyme employed in the subject methods andcompositions may also be administered by means of transforming patientcells with nucleic acids encoding the recombinant α-L-iduronidase. Thenucleic acid sequence so encoded may be incorporated into a vector fortransformation into cells of the subject to be treated. Preferredembodiments of such vectors are described herein. The vector may bedesigned so as to integrate into the chromosomes of the subject, e.g.,retroviral vectors, or to replicate autonomously in the host cells.Vectors containing encoding α-L-iduronidase nucleotide sequences may bedesigned so as to provide for continuous or regulated expression of theenzyme. Additionally, the genetic vector encoding the enzyme may bedesigned so as to stably integrate into the cell genome or to only bepresent transiently. The general methodology of conventional genetictherapy may be applied to polynucleotide sequences encodingα-L-iduronidase. Conventional genetic therapy techniques have beenextensively reviewed. (Friedman, Science 244:1275-1281(1989); Ledley, J.Inherit. Metab. Dis. 13:587-616 (1990); Tososhev, et al., Curr OpinionsBiotech. 1:55-61 (1990)).

[0084] A particularly preferred method of administering the recombinantenzyme is intravenously. A particularly preferred composition comprisesrecombinant α-L-iduronidase, normal saline, phosphate buffer to maintainthe pH at about 5.8 and human albumin. These ingredients maybe providedin the following amounts: α-L-iduronidase 0.05-0.2 mg/mL or12,500-50,000 units per mL Sodium chloride solution 150 mM in an IV bag,50-250 cc total volume Sodium phosphate buffer 10-50 mM, pH 5.8 Humanalbumin 1 mg/mL

[0085] Composition of Recombinant Human α-L-Iduronidase (rhIDU,Aldurazyme ™) Drug Product Composition Name of Ingredient Concentrationper vial Function rhIDU 100 U/mL 3.07 mg Active ingredient (Range 80-150U/mL) NaCl 150 mM 46.5 mg Tonicity Modifier Sodium Phosphate 92 mM 67.3mg Buffer monobasic Sodium Phosphate 8 mM 11.3 mg Buffer dibasicPolysorbate 80 10 μg/mL 0.05 mg Stabilizer

[0086] The proposed commercial formulation for Aldurazyme™ is 100Units/mL (approximately 0.58 mg/mL) for recombinant humanα-L-lduronidase (rhIDQ), 100 mM sodium phosphate, 150 mM sodiumchloride, and 10 μM/mL polysorbate 80, pH of 5.8. The Phase I studyformula was identical to the Phase III study formula and proposedcommercial formulation with the exception that polysorbate 80 was addedas a stabilizer in the Phase III and commercial formula. This commercialformulation was also used in Good Laboratory Practice (GLP) toxicologystudies.

[0087] Polysorbate 80, at a concentration of 10 μM/mL was added to theformulation to act as a stabilizer. The change was implemented when therhIDU production process was scaled up and prompted by the observationof a fine precipitate in the vialed drug product and coincided with thechange from polypropylene vials to glass vials. Formulation studies havedemonstrated that polysorbate-20 (10 μM/mL) and polysorbate-80 (5 μM/mL)both minimized the formation of precipitates in vialed Aldurazyme™ evenafter forced agitation. The concentration of polysorbate-20 orpolysorbate-80 needed to minimize the formation of precipitates was 5μM/mL for polysorbate-80 and 10 μM/mL for polysorbate-20. Preliminarydata demonstrated that Aldurazyme™, when formulated with polysorbate 80at 10 μM/mL, retained activity when stored at 2-8° C. Polysorbate 80 waschosen over polysorbate 20 because it performed slightly better inpreventing precipitate formation and it is more commonly used inmarketed pharmaceutical product formulations. Polysorbate is known to beeffective against agitation-induced aggregation of proteins, and areview of the literature regarding the use of polysorbate 80 in chronicintravenous therapies found the proposed level to be included inAldurazyme™ (10 μM/mL) to be well below that used in otherpharmaceutical formulations (Bam, et al., J. Pharm. Sci. 87(12):1554-9(1998); Kreilgaard, et al., J. Pharm. Sci. 87(12):1597-603 (1998)). Thesafety and efficacy of the commercial formulation were assessed in GoodLaboratory Practice (GLP) toxicology studies as well as Phase III study.

[0088] The invention having been described, the following examples areoffered to illustrate the subject invention by way of illustration, notby way of limitation.

EXAMPLE 1 Producing Recombinant α-L-lduronidase

[0089] Standard techniques such as those described by Sambrook, et al(Molecular Cloning: A Laboratory Manual, 2^(nd) ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1987)) maybe used to clone cDNAencoding human α-L-iduronidase. The human α-L-iduronidase cDNApreviously cloned was subcloned into PRCCMV (In Vitrogen) as aHindIII-XbaI fragment from a bluescript KS subclone. An intron cassettederived from the murine immunoglobulin Cα intron between exons 2 and 3was constructed using PCR amplification of bases 788-1372 (Tucker, etal., Proc. Natl. Acad. Sci. USA 78: 7684-7688 (1991) of clone pRIR14.5(Kakkis, et al., Nucleic Acids Res. 16:7796 (1988)). The cassetteincluded 136 bp of the 3′ end of exon 2 and 242 bp of the 5′ end of exon3, which would remain in the properly spliced cDNA. No ATG sequences arepresent in the coding region of the intron cassette. The intron cassettewas cloned into the HindIII site 5′ of the α-L-iduronidase cDNA. The neogene was deleted by digestion with Xhol followed by recircularizing thevector to make pCMVhldu.

[0090] One vial of the working cell bank is thawed and placed in threeT225 flasks in DME/F12 or PF-CHO plus supplements, plus 5% FBS and 500μg/ml G418. After 2-5 days, the cells are passaged using trypsin-EDTA toa 1-liter spinner flask in the same medium for 2-5 days. The cells arethen transferred to two 3-liter spinner flasks for 2-5 days, followed byfour 8-liter spinner flasks for 2-5 days. The inoculum from the 8-literspinner flasks is added to two 110-liter Applikon® stirred tankbioreactors with an 80-90 liter working volume. Macroporous cellulosemicrocarriers are added at 2 grams per liter (160 grams), with PF-CHO orDME/F12 plus supplements, 5% FBS and 500 μg/ml of G418 at a final volumeof 80-90 liters. The flask is stirred by an overhead drive with a marineimpeller. The culture is monitored for agitation speed, temperature, DOand pH probes and controlled the Applikon(® control system with a PCinterface. The parameters are controlled at the set points or range,35-37° C. depending on culture conditions, 40% air saturation, and pH6.95, using a heating blanket, oxygen sparger and base pump. The cultureis incubated for 3-5 days at which time the culture is emerging from thelog phase growth at 1-3×10⁶ cells per ml. Thereafter, perfusion isinitiated at an increasing rate with PF-CHO medium (with custommodifications, JRH Biosciences). The first four days of collection(range of 3-5 days) are set aside as “washout.” The collectionthereafter is the beginning of the production run. Production continueswith medium changes of as much as 2-3.5 culture volumes per day for20-36 days. The culture may be extended for 40 days or longer. Theculture is monitored for temperature, pH and DO on a continuous basis.The purification of the enzyme proceeds as described above. Collectedproduction medium containing iduronidase is then acidified to pH 5.3,filtered through a 0.2-micron filter and purified using Blue-Sepharosechromatography. The purified enzyme from multiple rounds ofBlue-Sepharose chromatography are then pooled and applied to a copperchelating column and eluted with glycerol in the buffer at a pH of 3.7.The enzyme is held at the acidic pH to inactivate potential viruses. Thecopper column eluate is then adjusted to pH 5.7 and 2 M NaCl and loadedon the phenyl Sepharose column. The enzyme is eluted at 0.7 M NaCl. Theeluate is concentrated and diafiltered into a formulation buffer of 150mM NaCl, 100 mM NaPO4, pH5.8. The enzyme is filtered through a 40 nMfilter to remove potential viruses and the filtrate adjusted to 0.001%polysorbate 80. The formulated enzyme is sterilely bulk filled intosterile polyethylene containers. The bulk enzyme is then filtered andfilled into 5 cc Type 1 glass vials appropriate for injectablepharmaceuticals, stoppered and capped.

EXAMPLE 2

[0091] For bioreactors using single cell suspensions, the seed train isprepared as described above in EXAMPLE 1. Using a single cell suspensionsimplifies bioreactor preparation and inoculation. The bioreactor isinoculated with cells in DMEM/F12 medium (25% of reactor volume) and JRH325 modified (25% of reactor volume). Medium equal to 50% of the workingreactor volume is added over 48 hours. Perfusion (and harvest) isstarted when cell density reaches 1.0 e⁶ and the perfusion medium is thesame as described above.

EXAMPLE 3

[0092] Short-term intravenous administration of purified humanrecombinant α-L-iduronidase to 9 MPS I dogs and 6 MPS I cats has shownsignificant uptake of an enzyme in a variety of tissues with anestimated 50% or more recovery in tissues 24 hours after a single dose.Although liver and spleen take up the largest amount of enzymes, andhave the best pathologic improvement, improvements in pathology andglycosaminoglycan content has been observed in many, but not alltissues. In particular, the cartilage, brain and heart valve did nothave significant improvement. Clinical improvement was observed in asingle dog on long-term treatment for 13 months, but other studies havebeen limited to 6 months or less. All dogs, and most cats, that receivedrecombinant human enzyme developed antibodies to the human product. TheIgG antibodies are of the complement activating type (probable canineIgG equivalent). This phenomena is also observed in at least 13% ofalglucerase-treated Gaucher patients. Proteinuria has been observed inone dog which may be related to immune complex disease. No other effectsof the antibodies have been observed in the other treated animals.Specific toxicity was not observed and clinical laboratory studies(complete blood counts, electrolytes, BLJN/creatinine, liver enzymes,urinalysis) have been otherwise normal.

[0093] Enzyme therapy at even small doses of 25,000 units (0.1 mg/kg/wk)resulted in significant enzyme distribution to some tissues anddecreases in GAG storage. If continued for over 1 year, significantclinical effects of the therapy were evident in terms of activity, sizeand overall appearance of health. The therapy at this dose did notimprove other tissues that are important sites for disease in thisentity such as cartilage and brain. Higher doses of 125,000 units (0.5mg/kg) given 5 times over two weeks demonstrate that improved tissuepenetration can be achieved and a therapeutic effect at the tissue levelwas accomplished in as little as 2 weeks. Studies at this increased doseare ongoing in two dogs for six months to date. These MPS I dogs areshowing significant clinical improvement and substantial decreases inurinary GAG excretion into the normal range. Other than an immunereaction controlled by altered administration techniques, the enzymetherapy has not shown significant clinical or biochemical toxicity.Enzyme therapy at this higher weekly dose is effective at improving someclinical features of MPS I and decreasing storage without significanttoxicity.

[0094] The results of these various studies in MPS I dogs and one studyin MPS I cats show that human recombinant α-L-iduronidase is safe.Although these same results provide significant rationale that thisrecombinant enzyme should be effective in treating α-L-iduronidasedeficiency, they do not predict the clinical benefits or the potentialimmunological risks of enzyme therapy in humans.

EXAMPLE 4

[0095] The human cDNA of α-L-iduronidase predicts a protein of 653 aminoacids and an expected molecular weight of 70,000 daltons after signalpeptide cleavage. Amino acid sequencing reveals alanine 26 at theN-terminus giving an expected protein of 629 amino acids. Humanrecombinant α-L-iduronidase has a Histidine at position 8 of the matureprotein. The predicted protein sequence comprises six potential N-linkedoligosaccharide modification sites. All of these sites are modified inthe recombinant protein. The third and sixth sites have beendemonstrated to contain one or more mannose 6-phosphate residuesresponsible for high affinity uptake into cells.

[0096] This peptide corresponds to Amnino Acids 26-45 of HumanRecombinant α-L-iduronidase with an N-terminus alanine and the followingsequence:

[0097]ala-glu-ala-pro-his-leu-val-his-val-asp-ala-ala-arg-ala-leu-trp-pro-leu-arg-arg(part of SEQ ID NO:2)

[0098] The recombinant enzyme has an apparent molecular weight of 82,000daltons on SDS-PAGE due to carbohydrate modifications. Purified humanrecombinant α-L-iduronidase has been sequenced by the UCLA ProteinSequencing facility. It is preferred to administer the recombinantenzyme intravenously. Human recombinant α-L-iduronidase was supplied forthe clinical trial in 10 mL polypropylene vials at a concentration of100,000-200,000 units per mL. The final dosage form of the enzyme usedin the clinical trial includes human recombinant α-L-iduronidase, normalsaline, and 100 mM phosphate buffer at pH 5.8. These are prepared in abag of normal saline. Polysorbate 80 at a final concentration of 0.001%was added to the formulation to stabilize the protein against shear,thereby avoiding precipitation in the final product vials. Final VialFormulation Currently in Use Component Composition α-L-iduronidaseTarget to 0.7 mg/mL or 100 (new) units per mL Sodium chloride solution150 mM Sodium phosphate buffer 100 mM, pH 5.8 Polysorbate 80 0.001%

[0099] Final Dosage Form Used in the Treatment of Patients ComponentComposition α-L-iduronidase product 5-12 fold dilution of vialconcentration Sodium chloride solution 50 mM Sodium phosphate buffer100-250 cc bag IV Human albumin 1 mg/ml

EXAMPLE 5

[0100] Phase I Study—Effects of Intravenous Administration ofα-L-Iduronidase in Patients with Mucopolysaccharidosis I (52 weeks)

[0101] Based on studies of cloning of cDNA encoding α-L-iduronidase(Scott, et al., Proc. Natl. Acad. Sci. USA 88: 9695-99 (1991);Stoltzfus, et al., J. Biol. Chem. 267: 6570-75 (1992)) and animalstudies showing effects of α-L-iduronidase to reduce lysosomal storagein many tissues (Shull, et al., Proc. Natl. Acad. Sci. USA 91: 12937-41(1994); Kakkis, et al., Biochem. Mol. Med. 58: 156-67 (1996)), a 52-weekstudy was conducted to assess the safety and clinical efficacy ofintravenous administration of highly purified α-L-iduronidase in tenpatients with mucopolysaccharidosis I (MPS I).

[0102] Recombinant human α-L-iduronidase was produced and purified togreater than 97-99%. Patients demonstrated typical clinicalmanifestations of the disorder and diagnosis was confirmed bybiochemical determination of α-L-iduronidase deficiency in leukocytes.

[0103] Patients were given recombinant human α-L-iduronidase (diluted innormal saline with 0.1% human serum albumin) intravenously at a dose of125,000 units per kg (using original assay and unit definition); 3,000units per kg were given over the first hour, and 61,000 units per kg ineach of the following two hours. The dose of 125,000 units per kg isequivalent to 100 SI units per kg using the new assay. The infusionswere prolonged up to 4-6 hours in patients who had hypersensitivityreactions.

[0104] At baseline and at 6, 12, 26 and 52 weeks depending on theevaluation, the patients underwent examinations including history,physical examinations by specialists, echocardiography, EKG, MRI,polysomnography (weeks 0 and 26), skeletal survey (weeks 0, 26, 52),range of motion measurements, corneal photographs, and skin biopsy (week0) to set up fibroblast cultures for enzyme determination andgenotyping. Range of motion measurements were performed with agoniometer and the maximum active (patient initiated) range was recordedfor each motion. Shoulder flexion is movement of the elbow anteriorlyfrom the side of the body and elbow and knee extension representstraightening of the joint. Degrees of restriction represent thedifference between the normal maximum range of motion for age and themeasured value. Polysomnography was performed according to AmericanThoracic Society guidelines and apneic events (cessation of oro-nasalairflow for 10 seconds or more), hypopneic events (decreased oro-nasalairflow of 50% or more with desaturation of 2% or more, or evidence ofarousal), minutes below 89% oxygen saturation and total sleep timerecorded among the standard measurements required. From these data anapnea/hypopnea index was calculated by dividing the total number ofapneic and hypopneic events by the number of hours of sleep. Biochemicalstudies included measurement of enzyme activity in leukocytes andbrushings of buccal mucosal, urinary glycosaminoglycan levels, and testsfor serum antibodies to recombinant human α-L-iduronidase (ELISA andWestern blot). Organ volumes were determined by analysis of MRI digitalimage data using Advantage Windows workstation software from GeneralElectric. The organ volume was measured in milliliters and was convertedto weight assuming a density of 1 gram per ml. Urinary glycosaminoglycanexcretion was assayed by an adaptation of a published method. Westernblots and ELISA assays for antibodies to recombinant humanα-L-iduronidase were performed by standard methods. Uronic acids andN-sulfate of urinary glycosaminoglycans were analyzed by the orcinol,carbazole and MBTH methods, and by electrophoretic separations.

[0105] All patients received weekly infusions of recombinant humanα-L-iduronidase administered for 52 weeks. The mean activity ofα-L-iduronidase in leukocytes was 0.04 units per mg before treatment andwhen measured on average 7 days after an infusion (i.e. immediatelybefore the next infusion), 4.98 units per mg, or 15.0 percent of normal.Enzyme activity was not detectable in buccal brushings prior totreatment, but 7 days after infusions it reached a level of 1 percent ofnormal.

[0106] Liver volume decreased by 19 to 37 percent from baseline in 9patients and 5 percent in one patient at 52 weeks; the mean decrease was25.0 percent (n=10, P<0.001). By 26 weeks, liver size was normal forbody weight and age in 8 patients (FIG. 1). In 2 patients (patients 6and 9) with the largest relative liver size at baseline, liver size wasclose to normal at 52 weeks (3.2 and 3.3 percent of body weight,respectively). Spleen size decreased in 8 patients by 13 to 42 percentfrom baseline (mean decrease of 20 percent in 10 patients, P<0.001).

[0107] Urinary glycosaminoglycan excretion declined rapidly by 3 to 4weeks and by 8-12 weeks had fallen by 60-80 percent of baseline. At 52weeks, the mean reduction was 63 percent (range 53-74; p<0.001). Eightof ten patients had a 75 percent or greater reduction of the baselineamount of urinary glycosaminoglycan in excess of the upper limit ofnormal for age. The results were confirmed by assay of uronic acids andN-sulfate (a test specific for heparan sulfate). Electrophoresis studiesof urine detected a significant reduction in heparan sulfate anddermatan sulfate excretion but some excess dermatan sulfate excretionpersisted in all patients.

[0108] The mean height increased 6.0 cm (5.2 percent) in the 6prepubertal patients (Table 2) and their mean height growth velocityincreased from 2.8 cm/yr to 5.2 cm/yr during treatment (P=0.011). Forall 10 patients, mean body weight increased 3.2 kg (8.8 percent) and themean increase was 4.2 kg (17.1 percent) for the 6 prepubertal patients(Table 2). In these 6 patients, the mean pretreatment weight growthvelocity increased from 1.7 kg per year to 3.8 kg per year duringtreatment (P=0.04).

[0109] Shoulder flexion (moving the elbow anteriorly) increased in 6 ofthe 8 subjects evaluated at baseline with a mean improvement for theright and left shoulders of 28° and 26°, respectively (P<0.002; FIG. 2).Elbow extension and knee extension increased by a mean of 7.0° (P<0.03)and 3.20 (P=0.10), respectively, in the 10 patients (FIG. 2).

[0110] Analysis of the improvement in individual patients revealed thatthe most restricted joints had the greatest improvement. For example atbaseline, patients 5, 9 and 10 could not flex their shoulders (move theelbow anteriorly) beyond 100°, which increased 21° to 51° aftertreatment. Similarly, patients 2 and 9 had a substantial increase inknee extension. The changes in range of motion were accompanied bypatient-reported increases in physical activities such as being able towash their hair, hold a hamburger normally, hang from monkey bars, andplay sports better.

[0111] Seven patients had a decrease in apnea and hypopnea events from155 to 60 per night upon treatment (a 61 percent decrease) with a changein mean apnea/hypopnea index (total number of events per hour) from 2.1to 1.0. Three patients had clinically significant sleep apnea and allthree improved during treatment. In patient 2, the apnea/hypopnea indexdecreased from 4.5 at baseline to 0.4 at 26 weeks and total time ofoxygen desaturation decreased from 48 minutes to 1 minute per night.Patient 6 required nightly continuous positive airway pressure therapybefore treatment due to severe desaturation (61 minutes below 89 percentsaturation with continuous positive airway pressure in 368 minutes ofsleep), but by 52 weeks, the patient tolerated the sleep study withoutCPAP and desaturated below 89 percent for only 8 minutes during 332minutes of sleep. Patient 9 had an apnea hypopnea index of 9.5 whichdecreased to 4.0 by 26 weeks. Patient 8 worsened with an apnea hypopneaindex of 0.1 increasing to 3.1 at 26 weeks and 9.3 at 52 weeks forunclear reasons. Eight of ten patients or their families reportedimproved breathing, and 5 of 7 noted quieter nighttime breathing,improved sleep quality and decreased daytime somnolence.

[0112] New York Heart Association functional classification wasdetermined by serial patient interviews. All 10 patients reportedimprovement by one or two classes but there was no significant objectivedata from echocardiographic studies to verify direct cardiac benefit.The improved functional scores may reflect improvements in other aspectsof MPS I disease rather than cardiac function. Comparing baseline to 52weeks of treatment, echocardiography demonstrated decreased tricuspidregurgitation or pulmonic regurgitation in 4 patients but two patients(patients 2 and 7) had worsening mitral regurgitation. At baseline,patient 6 had atrial flutter and clinical signs of cardiac failureincluding dyspnea at rest and peripheral edema. By 12 weeks, he hadnormal sinus rhythm with first degree block and his dyspnea at rest andpitting edema resolved.

[0113] All 10 patients reported a lack of endurance and limitations ofdaily activities before treatment but exercise tolerance was notformally tested. During treatment, all patients improved and by 26weeks, many were able to walk more, run and play sports. Patients 3, 4and 5 reported the resolution of severe incapacitating headaches aftertreatment for 6-12 weeks.

[0114] Several patients reported decreased photophobia or conjunctivalirritation. Visual acuity improved in one patient (20/1000 to 20/200 inone eye) and modestly in 2 others.

[0115] The results of this study indicate that intravenousadministration of the highly purified recombinant human α-L-iduronidaseof the present invention results in clinical and biochemical improvementin patients with Mucopolysaccharidosis I. The normalization of liversize and near normalization of urinary glycosaminoglycan excretion isconsistent with data from studies in dogs with Mucopolysaccharidosis I,which demonstrated clearance of storage in the liver and decreasedurinary glycosaminoglycan excretion in as little as 2 weeks.

[0116] Hypersensitivity reactions to the infusions of recombinant humanα-L-iduronidase were less severe than predicted from studies in dogs.Though important in some patients, recurrent urticaria was manageablewith premedication and adjustments in infusion rate. Antibodies specificto α-L-iduronidase were detected in 4 patients with usually subclinicalcomplement activation, and both the antibodies and complement activationdeclined with time. Similar IgG-mediated immune responses have beenpreviously noted in patients with Gaucher disease treat withglucocerebrosidase, although the events were more frequent in ourpatients. Mucopolysaccharidosis I patients with a null genotype may havea greater immune response than in these 10 patients, none of whom has anull.

[0117] Thus, recombinant human α-L-iduronidase can reduce lysosomalstorage and ameliorates some aspects of clinical disease ofMucopolysaccharidosis I.

EXAMPLE 6

[0118] Phase III Study—Effects of Intravenous Administration ofα-L-Iduronidase in Patients with Mucopolysaccharidosis 1 (26 weeks)

[0119] A multi-national, multi-center, double-blind, randomized,placebo-controlled study was conducted to further assess the safety andclinical efficacy of intravenous administration of highly purifiedα-L-Iduronidase in 45 MPS I patients.

[0120] Recombinant human α-L-Iduronidase was purified to greater than99%. The patients were characterized by age of at least five years old,less than 10 percent of normal enzyme activity, a baseline forced vitalcapacity (FVC) reflecting pulmonary function of 80% or less of percentpredicted normal, and a capability of standing for 6 minutes and walkingat least 5 meters. Of the 45 patients, 22 patients were treated withhighly purified α-L-Iduronidase and the remaining 23 received a placebo.Patients were administered human α-L-Iduronidase intravenously at a doseof 100 units per kilogram via a 4-hour intravenous infusion each weekfor 26 weeks.

[0121] Efficacy Endpoints

[0122] Patients were assessed by measuring primary efficacy endpoints,the change from baseline to week 26 in the % FVC and a six-minute walkdistance using the Wilcoxon Rank Sum Test. Patients were furtherassessed by secondary efficacy endpoints including apnea/hypoxia index(sleep study), liver organ volume (hepatomegaly), disability score index(Child Health Assessment Questionnaire/Health Assessment Questionnaire,CHAQ/HAQ), and shoulder flexion reflecting joint range of motion. Theseendpoints were measured as a change in baseline to week 26 by theAnalysis of Variance test. Patients were also assessed by measuringtertiary efficacy endpoints, including urinary glucosaminoglycan (GAG)levels, totally respiratory event index (sleep study), pain scale(CHAQ), shoulder extension, knee extension and flexion, quality of life(50-question Child Health Questionnaire Physical Functioning, CHQ PF 50;87-question Child Health Questionnaire directed to the child withquestions combined to create concepts, CHQ CF87; 36-question Short FormHealth Status Survey, SF-36), growth in prepubertal only, visual acuity,echocardiogram, force expriatory volume (FEVI), and investigator globalassessment. The investigator global assessment comprises a series ofseven categories in which the investigator is providing an assessment asto how each patient is improving during the trial.

[0123] Safety Endpoints

[0124] Safety was assessed by measurement of the frequency of adverseevents, serious adverse events, and infusion-associated reactions,immunogenicity testing, and measurement of other safety parameters byphysical examinations, testing of vital signs, brain/cranio-cervicaljunction magnetic resonance imaging (MRI, and standard laboratoryevaluations.

Results

[0125] Efficacy Endpoints

[0126] With respect to primary efficacy endpoints, a statisticallysignificant difference (p=0.028) was seen in the change in % predictedFVC (see Table I). A close to statistically significant difference(p=0.066) was noted in the change in 6-minute walk (Table II).

[0127] Although there was no significant overall difference observed inthe sleep apnea/hypopnea index, a reduction of events was observed inenzyme-treated patients with clinically significant disease (n=6/9,p=0.011). Consistent with the prior study, there was a significantreduction in liver volume (p=0.001) and hence improvement in occurrenceof hepatomegaly. There were no significant differences in CHAQ/HAQDiability Index or Joint Range of Motion, although there was a trendtowards improvement in more severe patients. There was a statisticallysignificant rapid reduction in urinary GAG (p<0.001). Trends in favor ofenzyme treatment were noted in measurements of right shoulder extension,left knee flexion, and LVDS (Left Venticle Internal Dimension atEnd-Systole in cm) as measured by echocardiography. TABLE I PercentPredicted Change From Baseline Intent To Treat Population DifferenceBaseline (% Week 26 (% from Predicted) Predicted) Change Placeboα-L-Iduronidase 48.4 ± 14.85 50.2 ± 17.10 1.8 ± 7.70 4.5 (Aldurazyme ™)p = 0.028 n = 22 Placebo 54.2 ± 16.00 51.5 ± 13.13 −2.7 ± 7.12   n = 23

[0128] TABLE II Six-Minute Walk Change from Baseline Intent To TreatPopulation Difference Baseline Week 26 from (m) (m) Change Placeboα-L-Idur- 319.0 ± 131.41 338.8 ± 127.06 19.7 ± 68.56 38.1 onidase p =0.066 (Aldura- zyme ™) n = 22 Placebo 366.7 ± 113.68 348.3 ± 128.81−18.3 ± 67.49   n = 23

[0129] Comparison with Phase I Study

[0130] The results from measurement of secondary and tertiary endpointswere consistent with that of the Phase I study. For example, in bothstudies there was a significant reduction in liver volume (p=0.001).Liver volume recovered to nomal in almost 60% of enzyme-treated patientsin the Phase III study at the end of 26 weeks. Similarly, in the Phase Istudy, liver size was normal for body weight and age in eight of tenpatients by 26 weeks. In both studies, there was a reduction in sleepapnea/hypopnea events. As described above, in the Phase In study, areduction in events (p=0.011) was observed in six of nine enzyme-treatedpatients with clinically significant disease. Similarly, seven of tenpatients in the Phase I study showed a decrease in apnea and hypopneaevents from 155 to 60 per night upon treatment with a reduction in themean apnea/hypopnea index. There was also an improvement in the jointrange of motion of more severe patients treated with the enzyme. UrinaryGAG excretion rapidly declined in enzyme-treated patients in bothstudies. In the Phase III study, there was a statistically significantrapid reduction in urinary GAG levels (p<0.001). Similarly, in the PhaseI study, urinary GAG excretion declined rapidly by three to four weeksand by eight to twelve weeks had fallen by 60-80% of baseline. Thus,there appeared to a strong correclations in the secondary and tertiaryefficacy endpoints of Phase I and III studies.

[0131] The results show that α-L-Iduronidase appears to be safe andwell-tolerated. The types of adverse events were similar between days ofinfusion and non-infusion days. The frequency of infusion—associatedreactions was similar between placbo and enzyme-treated groups. Withrespect to immunogenicity testing of IgG, all 22 patients in theenzyme-reated group seroconverted with a mean time to seroconversion of41 days. Seroconversion did not result in increased infusion-associatedreactions or other adverse events. Among three patients tested for IgE,including one patient from the placebo group and two enyme-treatedpatients, all IgE tests were negative. There were no clinicallysignificant changes in observations from physical examination, vital,and brain/cranio—cervical junction MRI from baseline to week 26.Standard laboratory evaluations showed: (1) no significant laboratorychnges indicating a negative treatment effect; (2) a significantincrease in platelet counts in enzyme—treated patients; and (3)improvement in liver enzyme abnormalities in enyme-treated patients.

[0132] Summary

[0133] In the Phase I studies i) all patients developed antibodies tothe treatment with all 10 to contaminating proteins and 4 to IDU itself;ii) 5 patients (50%) had clinical manifestations of an allergic responseof which the most common urticaria (hives); and iii) several of thesereactions were classified as serious adverse events (SAEs) (meaning theyrequired medical intervention) related to treatment withα-L-Iduronidase.

[0134] In the Phase III study, i) all patients developed antibodies tothe treatment but it is not yet known whether these were to CP or IDUitself; ii) clinical manifestations of an allergic response were mild inall patients and were comparable between the placebo and α-L-Iduronidasetreated groups; iii) there were no SAEs considered related to treatmentwith x-L-Iduronidase; and iv) there was no urticaria reported.

[0135] In summary, the efficacy data gathered in the MPS I dog studiesand the two human clinical trials tells a consistent story ofimprovement in disease symptoms. The safety profile of the productimproved significantly in the Phase im versus the Phase I. Thiscorroborates the theory that the material of increased purity used inthe Phase III trial is an improvment over the material used in the PhaseI trial.

[0136] The invention, and the manner and process of making and using it,are now described in such full, clear, concise and exact terms as toenable any person skilled in the art to which it pertains, to make anduse the same. It is to be understood that the foregoing describespreferred embodiments of the present invention and that modificationsmay be made therein without departing from the spirit or scope of thepresent invention as set forth in the claims. To particularly point outand distinctly claim the subject matter regarded as invention, thefollowing claims conclude this specification.

1 2 1 6200 DNA Homo sapiens CDS (1558)...(3510) 1 gacggatcgg gagatctcccgatcccctat ggtcgactct cagtacaatc tgctctgatg 60 ccgcatagtt aagccagtatctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120 cgagcaaaat ttaagctacaacaaggcaag gcttgaccga caattgcatg aagaatctgc 180 ttagggttag gcgttttgcgctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240 gattattgac tagttattaatagtaatcaa ttacggggtc attagttcat agcccatata 300 tggagttccg cgttacataacttacggtaa atggcccgcc tggctgaccg cccaacgacc 360 cccgcccatt gacgtcaataatgacgtatg ttcccatagt aacgccaata gggactttcc 420 attgacgtca atgggtggactatttacggt aaactgccca cttggcagta catcaagtgt 480 atcatatgcc aagtacgccccctattgacg tcaatgacgg taaatggccc gcctggcatt 540 atgcccagta catgaccttatgggactttc ctacttggca gtacatctac gtattagtca 600 tcgctattac catggtgatgcggttttggc agtacatcaa tgggcgtgga tagcggtttg 660 actcacgggg atttccaagtctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720 aaaatcaacg ggactttccaaaatgtcgta acaactccgc cccattgacg caaatgggcg 780 gtaggcgtgt acggtgggaggtctatataa gcagagctct ctggctaact agagaaccca 840 ctgcttaact ggcttatcgaaattaatacg actcactata gggagaccca agcttcgcag 900 aattcctgcg gctgctacagtgtgtccagc gtcctgcctg gctgtgctga gcgctggaac 960 agtggcgcat cattcaagtgcacagttacc catcctgagt ctggcacctt aactggcaca 1020 attgccaaag tcacaggtgagctcagatgc ataccaggac attgtatgac gttccctgct 1080 cacatgcctg ctttcttcctataatacaga tgctcaacta actgctcatg tccttatatc 1140 acagagggaa attggagctatctgaggaac tgcccagaag ggaagggcag aggggtcttg 1200 ctctccttgt ctgagccataactcttcttt ctaccttcca gtgaacacct tcccacccca 1260 ggtccacctg ctaccgccgccgtcggagga gctggccctg aatgagctct tgtccctgac 1320 atgcctggtg cgagctttcaaccctaaaga agtgctggtg cgatggctgc atggaaatga 1380 ggagctgtcc ccagaaagctacctagtgtt tgagccccta aaggagccag gcgagggagc 1440 caccacctac ctggtgacaagcgtgttgcg tgtatcagct gaaagcttga tatcgaattc 1500 cggaggcgga accggcagtgcagcccgaag ccccgcagtc cccgagcacg cgtggcc atg 1560 Met 1 cgt ccc ctg cgcccc cgc gcc gcg ctg ctg gcg ctc ctg gcc tcg ctc 1608 Arg Pro Leu Arg ProArg Ala Ala Leu Leu Ala Leu Leu Ala Ser Leu 5 10 15 ctg gcc gcg ccc ccggtg gcc ccg gcc gag gcc ccg cac ctg gtg cat 1656 Leu Ala Ala Pro Pro ValAla Pro Ala Glu Ala Pro His Leu Val His 20 25 30 gtg gac gcg gcc cgc gcgctg tgg ccc ctg cgg cgc ttc tgg agg agc 1704 Val Asp Ala Ala Arg Ala LeuTrp Pro Leu Arg Arg Phe Trp Arg Ser 35 40 45 aca ggc ttc tgc ccc ccg ctgcca cac agc cag gct gac cag tac gtg 1752 Thr Gly Phe Cys Pro Pro Leu ProHis Ser Gln Ala Asp Gln Tyr Val 50 55 60 65 ctc agc tgg gac cag cag ctcaac ctc gcc tat gtg ggc gcc gtc cct 1800 Leu Ser Trp Asp Gln Gln Leu AsnLeu Ala Tyr Val Gly Ala Val Pro 70 75 80 cac cgc ggc atc aag cag gtc cggacc cac tgg ctg ctg gag ctt gtc 1848 His Arg Gly Ile Lys Gln Val Arg ThrHis Trp Leu Leu Glu Leu Val 85 90 95 acc acc agg ggg tcc act gga cgg ggcctg agc tac aac ttc acc cac 1896 Thr Thr Arg Gly Ser Thr Gly Arg Gly LeuSer Tyr Asn Phe Thr His 100 105 110 ctg gac ggg tac ctg gac ctt ctc agggag aac cag ctc ggg ttt gag 1944 Leu Asp Gly Tyr Leu Asp Leu Leu Arg GluAsn Gln Leu Gly Phe Glu 115 120 125 ctg atg ggc agc gcc tcg ggc cac ttcact gac ttt gag gac aag cag 1992 Leu Met Gly Ser Ala Ser Gly His Phe ThrAsp Phe Glu Asp Lys Gln 130 135 140 145 cag gtg ttt gag tgg aag gac ttggtc tcc agc ctg gcc agg aga tac 2040 Gln Val Phe Glu Trp Lys Asp Leu ValSer Ser Leu Ala Arg Arg Tyr 150 155 160 atc ggt agg tac gga ctg gcg catgtt tcc aag tgg aac ttc gag acg 2088 Ile Gly Arg Tyr Gly Leu Ala His ValSer Lys Trp Asn Phe Glu Thr 165 170 175 tgg aat gag cca gac cac cac gacttt gac aac gtc tcc atg acc atg 2136 Trp Asn Glu Pro Asp His His Asp PheAsp Asn Val Ser Met Thr Met 180 185 190 caa ggc ttc ctg aac tac tac gatgcc tgc tcg gag ggt ctg cgc gcc 2184 Gln Gly Phe Leu Asn Tyr Tyr Asp AlaCys Ser Glu Gly Leu Arg Ala 195 200 205 gcc agc ccc gcc ctg cgg ctg ggaggc ccc ggc gac tcc ttc cac acc 2232 Ala Ser Pro Ala Leu Arg Leu Gly GlyPro Gly Asp Ser Phe His Thr 210 215 220 225 cca ccg cga tcc ccg ctg agctgg ggc ctc ctg cgc cac tgc cac gac 2280 Pro Pro Arg Ser Pro Leu Ser TrpGly Leu Leu Arg His Cys His Asp 230 235 240 ggt acc aac ttc ttc act ggggag gcg ggc gtg cgg ctg gac tac atc 2328 Gly Thr Asn Phe Phe Thr Gly GluAla Gly Val Arg Leu Asp Tyr Ile 245 250 255 tcc ctc cac agg aag ggt gcgcgc agc tcc atc tcc atc ctg gag cag 2376 Ser Leu His Arg Lys Gly Ala ArgSer Ser Ile Ser Ile Leu Glu Gln 260 265 270 gag aag gtc gtc gcg cag cagatc cgg cag ctc ttc ccc aag ttc gcg 2424 Glu Lys Val Val Ala Gln Gln IleArg Gln Leu Phe Pro Lys Phe Ala 275 280 285 gac acc ccc att tac aac gacgag gcg gac ccg ctg gtg ggc tgg tcc 2472 Asp Thr Pro Ile Tyr Asn Asp GluAla Asp Pro Leu Val Gly Trp Ser 290 295 300 305 ctg cca cag ccg tgg agggcg gac gtg acc tac gcg gcc atg gtg gtg 2520 Leu Pro Gln Pro Trp Arg AlaAsp Val Thr Tyr Ala Ala Met Val Val 310 315 320 aag gtc atc gcg cag catcag aac ctg cta ctg gcc aac acc acc tcc 2568 Lys Val Ile Ala Gln His GlnAsn Leu Leu Leu Ala Asn Thr Thr Ser 325 330 335 gcc ttc ccc tac gcg ctcctg agc aac gac aat gcc ttc ctg agc tac 2616 Ala Phe Pro Tyr Ala Leu LeuSer Asn Asp Asn Ala Phe Leu Ser Tyr 340 345 350 cac ccg cac ccc ttc gcgcag cgc acg ctc acc gcg cgc ttc cag gtc 2664 His Pro His Pro Phe Ala GlnArg Thr Leu Thr Ala Arg Phe Gln Val 355 360 365 aac aac acc cgc ccg ccgcac gtg cag ctg ttg cgc aag ccg gtg ctc 2712 Asn Asn Thr Arg Pro Pro HisVal Gln Leu Leu Arg Lys Pro Val Leu 370 375 380 385 acg gcc atg ggg ctgctg gcg ctg ctg gat gag gag cag ctc tgg gcc 2760 Thr Ala Met Gly Leu LeuAla Leu Leu Asp Glu Glu Gln Leu Trp Ala 390 395 400 gaa gtg tcg cag gccggg acc gtc ctg gac agc aac cac acg gtg ggc 2808 Glu Val Ser Gln Ala GlyThr Val Leu Asp Ser Asn His Thr Val Gly 405 410 415 gtc ctg gcc agc gcccac cgc ccc cag ggc ccg gcc gac gcc tgg cgc 2856 Val Leu Ala Ser Ala HisArg Pro Gln Gly Pro Ala Asp Ala Trp Arg 420 425 430 gcc gcg gtg ctg atctac gcg agc gac gac acc cgc gcc cac ccc aac 2904 Ala Ala Val Leu Ile TyrAla Ser Asp Asp Thr Arg Ala His Pro Asn 435 440 445 cgc agc gtc gcg gtgacc ctg cgg ctg cgc ggg gtg ccc ccc ggc ccg 2952 Arg Ser Val Ala Val ThrLeu Arg Leu Arg Gly Val Pro Pro Gly Pro 450 455 460 465 ggc ctg gtc tacgtc acg cgc tac ctg gac aac ggg ctc tgc agc ccc 3000 Gly Leu Val Tyr ValThr Arg Tyr Leu Asp Asn Gly Leu Cys Ser Pro 470 475 480 gac ggc gag tggcgg cgc ctg ggc cgg ccc gtc ttc ccc acg gca gag 3048 Asp Gly Glu Trp ArgArg Leu Gly Arg Pro Val Phe Pro Thr Ala Glu 485 490 495 cag ttc cgg cgctag cgc gcg gct gag gac ccg gtg gcc gcg gcg ccc 3096 Gln Phe Arg Arg *Arg Ala Ala Glu Asp Pro Val Ala Ala Ala Pro 500 505 510 cgc ccc tta cccgcc ggc ggc cgc ctg agg ctg cgc ccc gcg ctg cgg 3144 Arg Pro Leu Pro AlaGly Gly Arg Leu Arg Leu Arg Pro Ala Leu Arg 515 520 525 ctg ccg tcg cttttg ctg gtg cac gtg tgt gcg cgc ccc gag aag ccg 3192 Leu Pro Ser Leu LeuLeu Val His Val Cys Ala Arg Pro Glu Lys Pro 530 535 540 ccc ggg cag gtcacg cgg ctc cgc gcc ctg ccc ctg acc caa ggg cag 3240 Pro Gly Gln Val ThrArg Leu Arg Ala Leu Pro Leu Thr Gln Gly Gln 545 550 555 560 ctg gtt ctggtc tgg tcg gat gaa cac gtg ggc tcc aag tgc ctg tgg 3288 Leu Val Leu ValTrp Ser Asp Glu His Val Gly Ser Lys Cys Leu Trp 565 570 575 aca tac gagatc cag ttc tct cag gac ggt aag gcg tac acc ccg gtc 3336 Thr Tyr Glu IleGln Phe Ser Gln Asp Gly Lys Ala Tyr Thr Pro Val 580 585 590 agc agg aagcca tcg acc ttc aac ctc ttt gtg ttc agc cca gac aca 3384 Ser Arg Lys ProSer Thr Phe Asn Leu Phe Val Phe Ser Pro Asp Thr 595 600 605 ggt gct gtctct ggc tcc tac cga gtt cga gcc ctg gac tac tgg gcc 3432 Gly Ala Val SerGly Ser Tyr Arg Val Arg Ala Leu Asp Tyr Trp Ala 610 615 620 cga cca ggcccc ttc tcg gac cct gtg ccg tac ctg gag gtc cct gtg 3480 Arg Pro Gly ProPhe Ser Asp Pro Val Pro Tyr Leu Glu Val Pro Val 625 630 635 640 cca agaggg ccc cca tcc ccg ggc aat cca tgagcctgtg ctgagcccca 3530 Pro Arg GlyPro Pro Ser Pro Gly Asn Pro 645 650 gtgggttgca cctccaccgg cagtcagcgagctggggctg cactgtgccc atgctgccct 3590 cccatcaccc cctttgcaat atatttttatattttaaaaa aaaaaaaaaa aaaaaaaaaa 3650 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaag aattcctgca 3710 gcccggggga tccactagtt ctagagggcccgtttaaacc cgctgatcag cctcgactgt 3770 gccttctagt tgccagccat ctgttgtttgcccctccccc gtgccttcct tgaccctgga 3830 aggtgccact cccactgtcc tttcctaataaaatgaggaa attgcatcgc attgtctgag 3890 taggtgtcat tctattctgg ggggtggggtggggcaggac agcaaggggg aggattggga 3950 agacaatagc aggcatgctg gggatgcggtgggctctatg gcttctgagg cggaaagaac 4010 cagctggggc tcgagagctt ggcgtaatcatggtcatagc tgtttcctgt gtgaaattgt 4070 tatccgctca caattccaca caacatacgagccggaagca taaagtgtaa agcctggggt 4130 gcctaatgag tgagctaact cacattaattgcgttgcgct cactgcccgc tttccagtcg 4190 ggaaacctgt cgtgccagct gcattaatgaatcggccaac gcgcggggag aggcggtttg 4250 cgtattgggc gctcttccgc ttcctcgctcactgactcgc tgcgctcggt cgttcggctg 4310 cggcgagcgg tatcagctca ctcaaaggcggtaatacggt tatccacaga atcaggggat 4370 aacgcaggaa agaacatgtg agcaaaaggccagcaaaagg ccaggaaccg taaaaaggcc 4430 gcgttgctgg cgtttttcca taggctccgcccccctgacg agcatcacaa aaatcgacgc 4490 tcaagtcaga ggtggcgaaa cccgacaggactataaagat accaggcgtt tccccctgga 4550 agctccctcg tgcgctctcc tgttccgaccctgccgctta ccggatacct gtccgccttt 4610 ctcccttcgg gaagcgtggc gctttctcaatgctcacgct gtaggtatct cagttcggtg 4670 taggtcgttc gctccaagct gggctgtgtgcacgaacccc ccgttcagcc cgaccgctgc 4730 gccttatccg gtaactatcg tcttgagtccaacccggtaa gacacgactt atcgccactg 4790 gcagcagcca ctggtaacag gattagcagagcgaggtatg taggcggtgc tacagagttc 4850 ttgaagtggt ggcctaacta cggctacactagaaggacag tatttggtat ctgcgctctg 4910 ctgaagccag ttaccttcgg aaaaagagttggtagctctt gatccggcaa acaaaccacc 4970 gctggtagcg gtggtttttt tgtttgcaagcagcagatta cgcgcagaaa aaaaggatct 5030 caagaagatc ctttgatctt ttctacggggtctgacgctc agtggaacga aaactcacgt 5090 taagggattt tggtcatgag attatcaaaaaggatcttca cctagatcct tttaaattaa 5150 aaatgaagtt ttaaatcaat ctaaagtatatatgagtaaa cttggtctga cagttaccaa 5210 tgcttaatca gtgaggcacc tatctcagcgatctgtctat ttcgttcatc catagttgcc 5270 tgactccccg tcgtgtagat aactacgatacgggagggct taccatctgg ccccagtgct 5330 gcaatgatac cgcgagaccc acgctcaccggctccagatt tatcagcaat aaaccagcca 5390 gccggaaggg ccgagcgcag aagtggtcctgcaactttat ccgcctccat ccagtctatt 5450 aattgttgcc gggaagctag agtaagtagttcgccagtta atagtttgcg caacgttgtt 5510 gccattgcta caggcatcgt ggtgtcacgctcgtcgtttg gtatggcttc attcagctcc 5570 ggttcccaac gatcaaggcg agttacatgatcccccatgt tgtgcaaaaa agcggttagc 5630 tccttcggtc ctccgatcgt tgtcagaagtaagttggccg cagtgttatc actcatggtt 5690 atggcagcac tgcataattc tcttactgtcatgccatccg taagatgctt ttctgtgact 5750 ggtgagtact caaccaagtc attctgagaatagtgtatgc ggcgaccgag ttgctcttgc 5810 ccggcgtcaa tacgggataa taccgcgccacatagcagaa ctttaaaagt gctcatcatt 5870 ggaaaacgtt cttcggggcg aaaactctcaaggatcttac cgctgttgag atccagttcg 5930 atgtaaccca ctcgtgcacc caactgatcttcagcatctt ttactttcac cagcgtttct 5990 gggtgagcaa aaacaggaag gcaaaatgccgcaaaaaagg gaataagggc gacacggaaa 6050 tgttgaatac tcatactctt cctttttcaatattattgaa gcatttatca gggttattgt 6110 ctcatgagcg gatacatatt tgaatgtatttagaaaaata aacaaatagg ggttccgcgc 6170 acatttcccc gaaaagtgcc acctgacgtc6200 2 650 PRT Homo sapiens 2 Met Arg Pro Leu Arg Pro Arg Ala Ala LeuLeu Ala Leu Leu Ala Ser 1 5 10 15 Leu Leu Ala Ala Pro Pro Val Ala ProAla Glu Ala Pro His Leu Val 20 25 30 His Val Asp Ala Ala Arg Ala Leu TrpPro Leu Arg Arg Phe Trp Arg 35 40 45 Ser Thr Gly Phe Cys Pro Pro Leu ProHis Ser Gln Ala Asp Gln Tyr 50 55 60 Val Leu Ser Trp Asp Gln Gln Leu AsnLeu Ala Tyr Val Gly Ala Val 65 70 75 80 Pro His Arg Gly Ile Lys Gln ValArg Thr His Trp Leu Leu Glu Leu 85 90 95 Val Thr Thr Arg Gly Ser Thr GlyArg Gly Leu Ser Tyr Asn Phe Thr 100 105 110 His Leu Asp Gly Tyr Leu AspLeu Leu Arg Glu Asn Gln Leu Gly Phe 115 120 125 Glu Leu Met Gly Ser AlaSer Gly His Phe Thr Asp Phe Glu Asp Lys 130 135 140 Gln Gln Val Phe GluTrp Lys Asp Leu Val Ser Ser Leu Ala Arg Arg 145 150 155 160 Tyr Ile GlyArg Tyr Gly Leu Ala His Val Ser Lys Trp Asn Phe Glu 165 170 175 Thr TrpAsn Glu Pro Asp His His Asp Phe Asp Asn Val Ser Met Thr 180 185 190 MetGln Gly Phe Leu Asn Tyr Tyr Asp Ala Cys Ser Glu Gly Leu Arg 195 200 205Ala Ala Ser Pro Ala Leu Arg Leu Gly Gly Pro Gly Asp Ser Phe His 210 215220 Thr Pro Pro Arg Ser Pro Leu Ser Trp Gly Leu Leu Arg His Cys His 225230 235 240 Asp Gly Thr Asn Phe Phe Thr Gly Glu Ala Gly Val Arg Leu AspTyr 245 250 255 Ile Ser Leu His Arg Lys Gly Ala Arg Ser Ser Ile Ser IleLeu Glu 260 265 270 Gln Glu Lys Val Val Ala Gln Gln Ile Arg Gln Leu PhePro Lys Phe 275 280 285 Ala Asp Thr Pro Ile Tyr Asn Asp Glu Ala Asp ProLeu Val Gly Trp 290 295 300 Ser Leu Pro Gln Pro Trp Arg Ala Asp Val ThrTyr Ala Ala Met Val 305 310 315 320 Val Lys Val Ile Ala Gln His Gln AsnLeu Leu Leu Ala Asn Thr Thr 325 330 335 Ser Ala Phe Pro Tyr Ala Leu LeuSer Asn Asp Asn Ala Phe Leu Ser 340 345 350 Tyr His Pro His Pro Phe AlaGln Arg Thr Leu Thr Ala Arg Phe Gln 355 360 365 Val Asn Asn Thr Arg ProPro His Val Gln Leu Leu Arg Lys Pro Val 370 375 380 Leu Thr Ala Met GlyLeu Leu Ala Leu Leu Asp Glu Glu Gln Leu Trp 385 390 395 400 Ala Glu ValSer Gln Ala Gly Thr Val Leu Asp Ser Asn His Thr Val 405 410 415 Gly ValLeu Ala Ser Ala His Arg Pro Gln Gly Pro Ala Asp Ala Trp 420 425 430 ArgAla Ala Val Leu Ile Tyr Ala Ser Asp Asp Thr Arg Ala His Pro 435 440 445Asn Arg Ser Val Ala Val Thr Leu Arg Leu Arg Gly Val Pro Pro Gly 450 455460 Pro Gly Leu Val Tyr Val Thr Arg Tyr Leu Asp Asn Gly Leu Cys Ser 465470 475 480 Pro Asp Gly Glu Trp Arg Arg Leu Gly Arg Pro Val Phe Pro ThrAla 485 490 495 Glu Gln Phe Arg Arg Arg Ala Ala Glu Asp Pro Val Ala AlaAla Pro 500 505 510 Arg Pro Leu Pro Ala Gly Gly Arg Leu Arg Leu Arg ProAla Leu Arg 515 520 525 Leu Pro Ser Leu Leu Leu Val His Val Cys Ala ArgPro Glu Lys Pro 530 535 540 Pro Gly Gln Val Thr Arg Leu Arg Ala Leu ProLeu Thr Gln Gly Gln 545 550 555 560 Leu Val Leu Val Trp Ser Asp Glu HisVal Gly Ser Lys Cys Leu Trp 565 570 575 Thr Tyr Glu Ile Gln Phe Ser GlnAsp Gly Lys Ala Tyr Thr Pro Val 580 585 590 Ser Arg Lys Pro Ser Thr PheAsn Leu Phe Val Phe Ser Pro Asp Thr 595 600 605 Gly Ala Val Ser Gly SerTyr Arg Val Arg Ala Leu Asp Tyr Trp Ala 610 615 620 Arg Pro Gly Pro PheSer Asp Pro Val Pro Tyr Leu Glu Val Pro Val 625 630 635 640 Pro Arg GlyPro Pro Ser Pro Gly Asn Pro 645 650

What is claimed is:
 1. A recombinant α-L-iduronidase enzyme orbiologically active fragments or mutein thereof with a purity of greaterthan 99%.
 2. The recombinant α-L-iduronidase enzyme or biologicallyactive fragments or mutein thereof of claim 1 with a specific activitygreater than about 240,000 units per milligram protein.
 3. A method ofproducing human α-L-iduronidase of claim 1, comprising the steps of: (a)preparing a seed train of cells transformed with nucleic acids encodingfor inoculation into a bioreactor; (b) preparing a mixture containingmacroporous microcarriers by washing and autoclaving said microcarriersin phosphate buffered saline, combining said microcarriers with growthmedium and fetal bovine serum, and pumping said microcarrier mixtureinto said bioreactor; (c) inoculating and incubating said cells in saidbioreactor under control of pH, dissolved oxygen and perfusion; and (d)harvesting cells when cell density reaches about 10⁶.
 4. A method ofpreparing seed train of said cells of claim 3 for mass production,comprising: (a) washing and resuspending an aliquot of working cell bankCHO cells 2.131 in culture medium containing protein-free medium withsupplementation of 7.6 mg/L thymidine, 13.6 mg/L hypoxanthine, 375 μg/mLG418 and 5% fetal bovine serum; (b) incubating said cell suspension fortwo to three days at 37° C. and 5% carbon dioxide in three 225cm-flasks; (c) splitting said cell suspension by adding the cellssequentially to one 1-liter spinner flask, two 3-liter flasks, and four8-liter flasks; (d) rotating said cell suspension at 50 revolutions perminute, followed by increasing inoculum volume by incubating andsubculturing cells to a final cell density of about 2.0×10⁵ to 2.5×10⁵.5. A method of purifying of α-L-iduronidase of claim 1 to greater thanabout 99% purity, comprising the steps of: (a) harvesting and filteringfluid obtained from a culture of cells transformed with nucleic acidsencoding said human α-L-iduronidase; (b) adjusting the pH of the fluidto an acidic pH, followed by filtration through a 0.2 micron to 0.54micron filter; (c) passing the fluid through a blue sepharose FF columnto capture said human recombinant α-L-iduronidase; (d) passing the fluidthrough a copper chelating sepharose column to remove contaminating CHOproteins; (e) passing the fluid through a phenyl sepharose column toreduce residual leached Cibacron blue dye and copper ions carried overfrom previous columns; and (f) concentrating and diafiltering thepurified α-L-iduronidase.
 6. The method of claim 5, wherein said bluesepharose FF column is used to purify said human α-L-iduronidase sevento ten fold.
 7. The method of claim 5, wherein said method comprisesusing 10% glycerol in all buffers to increase the quantitative recoveryof said human α-L-iduronidase.